Perfluorooctanoic Acid Treatment Research Articles

PFOA Exposure Prior to Hepatocyte Differentiation Leads to Gene Expression Changes Implicated in Non-Alcoholic Fatty Liver Disease

Background: Perfluorooctanoic acid (PFOA), is a persistent fluorinated compound with oil and water repelling properties found in cookware, food packaging and municipal water systems. Adult animals exposed to PFOA develop hepatomegaly, fatty liver, peroxisome proliferation, and immunotoxicity. Rodents exposed to PFCs in utero have altered hepatic lipid metabolism, increased hepatic de novo lipogenesis and susceptibility to non-alcoholic fatty liver disease (NAFLD), but underlying molecular mechanisms remain unknown. With increasing rates of obesity, diabetes, and NAFLD it is critical to examine the mechanisms by which in utero exposure to PFOA contributes to the development of metabolic syndrome in offspring. Objective: To determine mechanism by which PFOA alters gene expression in undifferentiated hepatic progenitor cells. Design/methods: HepaRG cells, a human derived hepatocyte progenitor cell line, was treated with 0.5uM PFOA or vehicle for 48 hours followed by differentiation into hepatocytes. Total RNA was extracted using the RNeasy (Qiagen) [total RNA A260/280>2 and RNA integrity number >7 (Agilent Bioanalyzer)] to generate libraries with the Illumina TruSeq stranded total RNA kit. RNA-Seq was performed using 85 bp single-end read sequencing to generate >20 million reads per sample. RNAseq data was aligned to hg38 using STAR v2.6.1a and then quantified with featureCounts v1.6.2. DESeq2 identified differentially expressed genes via FDR (false discovery rate) after Bonferroni correction. Differentially expressed gene lists were used for Ingenuity Pathway Analysis (IPA) to identify pathways of biological significance.Results: PFOA treatment resulted in increased expression of transcription factors EGR1 (early growth response protein 1), NR4A1 (nuclear receptor Nur77), EGR2 (early growth response protein 2), KLF10 (Krueppel-like factor 10) and FOSL1 (Fos-related antigen 1), key genes linked to impaired hepatic insulin signaling, hepatic lipid metabolism, steatosis and fibrosis (fold change > 1.5; q <0.05). IPA identified enrichment of canonical pathways with biological relevance including hepatic fibrosis signaling, stellate cell activation, VDR/RXR/TR activation, and Type 2 diabetes mellitus signaling (p<0.01). Conclusion: Hepatocyte progenitor cells exposed to low dose PFOA for 48 hours prior to differentiation results in changes in expression of key metabolic genes linked to the development of NAFLD and enrichment of biologically relevant pathways associated with hepatic fibrosis and hepatocellular carcinoma. These results suggest that PFOA exposure in utero may have lasting effects on hepatic glucose and lipid metabolism after differentiation. Further studies are needed to characterize the longstanding metabolic effects of in utero PFOA exposure in offspring and the mechanisms driving the persistence of these changes.

Insights into the degradation mechanism of perfluorooctanoic acid under visible-light irradiation through fabricating flower-shaped Bi5O7I/ZnO n-n heterojunction microspheres

• Bi 5 O 7 I/ZnO n-n heterojunction was facilely prepared by in-situ phase transition. • n-n Bi 5 O 7 I/ZnO efficiently degraded PFOA under visible light irradiation. • The experimental mechanism was verified by DFT calculation. • h + were the active substance responsible for PFOA degradation. Perfluorooctanoic acid (PFOA), which is an emerging contaminant, has received extensive attention in recent times due to its high toxicity and environmental risk. In this study, BiOI supported on Zn-Al hydrotalcite (BOI 0.04 -BHZA) was calcined at 400 ℃ to obtain flower-shaped Bi 5 O 7 I/ZnO n-n heterojunction microspheres for the photocatalytic degradation of PFOA under visible light irradiation. The samples were characterized by X-ray diffraction, Fourier transform infrared, UV–vis diffuse reflectance spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. Compared with BOI (k 1 = 0.0044 h −1 ), BHZA (k 2 = 0.0054 h −1 ), and BOI 0.04 -BHZA (k 3 = 0.0073 h −1 ), the degradation rate constant of Bi 5 O 7 I/ZnO n-n heterojunction microspheres (k 4 = 0.013 h −1 ) increased by 2.9, 2.4, and 1.8 times, respectively. Approximately 91% of PFOA was degraded after 6 h of irradiation. The excellent photocatalytic performance was ascribed to the Bi 5 O 7 I/ZnO n-n heterojunction formed by calcination, which enlarged the photoresponse to the visible light region and increased the separation efficiency of electron-hole pairs. Moreover, the degradation pathway of PFOA was investigated using high-performance liquid chromatography-mass spectrometry and ion chromatography assisted by density functional theory calculations. The results demonstrated that the carboxylic groups of PFOA was vulnerable to attack by the photoproduction hole. The formed unstable perfluoroheptyl radicals transformed into shorter chain perfluorocarboxylic acids by the elimination of CF 2 units. It was expected that this Bi 5 O 7 I/ZnO n-n heterojunction photocatalyst would be a promising candidate for PFOA treatment.

Perfluorooctanoic acid inhibits the maturation rate of mouse oocytes cultured in vitro by triggering mitochondrial and DNA damage.

Perfluorooctanoic acid (PFOA) is widely used in the manufacture of household and industrial products. It has certain toxicity and leaves many residues in the environment. Numerous studies have shown that PFOA exhibits endocrine disrupting properties and immunotoxicity and induces developmental defects. However, there is very little information regarding its toxicity on oocytes. We cultured denuded oocytes in maturation medium supplemented with 0, 300, or 500 PFOA during IVM and evaluated the maturation of oocytes from the aspects of ROS(DCFH-DA), mitochondria(MitoOrange and JC-1), DNA damage(P-H2AX), and cytoskeleton(β-tubulin). Compared with the control group, the PFOA treatment group exhibited significantly reduced proportion of oocytes matutation. Furthermore, the DCFH-DA test showed that PFOA significantly increased reactive oxygen species (ROS) levels. PFOA disrupted mitochondrial distribution and decreased mitochondrial function as assessed using MitoOrange and JC-1. In addition, PFOA-treated oocytes exhibited a significantly higher percentage of P-H2AX, defective β-tubulin, abnormal chromosome alignment, lower expression of the anti-apoptotic gene Bcl-2, and higher expression of the apoptotic genes caspase3 and Bax. In summary, PFOA could negatively and directly affect oocyte maturation in vitro and cause oxidative stress, mitochondrial function disruption, DNA damage, cytoskeleton damage, and apoptosis.

Perfluorooctanoic acid-induced immunotoxicity via NF-kappa B pathway in zebrafish (Danio rerio) kidney

Perfluorooctanoic acid (PFOA) is widely used in industrial production due to its stable chemical structure and hydrophobic and oleophobic characteristics. PFOA has been frequently detected in environmental media and organisms, leading to increased health risks. There is a lack of information about the immunotoxicity of aquatic organisms induced by PFOA, and the molecular mechanisms remain unclear. In this study, LC-MS analysis proved that PFOA can accumulate in the kidney of zebrafish. In the 0.05 mg/L PFOA treatment group, the accumulation of PFOA in the kidney after 21 days of exposure significantly increased by 79.89%, compared to 14 days of exposure. And a hydropic endoplasmic reticulum, swelling of mitochondria and vacuolization were observed in kidney immune cells of zebrafish. The Toll-like receptor 2 (TLR2)/myeloid differentiation factor 88 (myd88)/NF-κB (P65) pathway was activated when PFOA exerted its effects, which led to regulation of antibody expression; RT-PCR results showed that the mRNA expression level of interleukin-4 (IL-4) decreased in a dose-dependent manner, decreasing to 29.6% of the control level in the 1 mg/L PFOA group after 21 d of exposure. According to triangle plot analysis, immunoglobulin exhibited a notable stress response to PFOA at an early phase; a high concentration of PFOA may disrupt the immune system of zebrafish. Third-order polynomial fitting analysis showed that the high-mRNA-expression regions of IL-4 and antibodies were partially consistent. The results indicated that PFOA could affect antibodies by increasing the concentrations of proinflammatory cytokines. Changes in antibody levels further influenced the expression of other cytokines, which eventually caused disorders in the zebrafish immune system. This study expands the understanding of PFOA-induced immunosuppression and suggests that toxicity mechanisms should be considered for further health risk assessment of emerging pollutants.

Perfluorooctanoic acid in indoor particulate matter triggers oxidative stress and inflammation in corneal and retinal cells

To investigate the particle size distribution of particulate matter and the concentration of specific perfluorinated compounds in indoor dust samples from several locations. Then, we used cell-based assays to investigate the effect of perfluorinated compounds on human corneal epithelial (HCEpiC), endothelial cells (HCEC) and retinal pigment epithelial cells (RPE). Indoor dust samples were collected at five different locations and PM50–10, PM10–2.5, and PM2.5–1 were fractionized. The presence and levels of 8:2 fluorotelomer alcohol, 10:2 fluorotelomer alcohol, and perfluorooctanoic acid were detected by gas chromatography–mass spectrometry. The effect of perfluorooctanoic acid on the activation of reactive oxygen species, transepithelial resistance as well as the expression of interleukin (IL)-6 and IL-8 were determined. The basolateral media of human corneal epithelial or human corneal endothelial cells were used to treat human corneal endothelial or retinal pigment epithelial cells, respectively to indicate the potential of ocular surface inflammation may result in retinal inflammation. Among perfluorinated compounds, only perfluorooctanoic acid was detected in all indoor dust samples. Perfluorooctanoic acid had the highest concentration among all perfluorinated compounds in the samples. Exposure to perfluorooctanoic acid impaired tight junction sealing and increased the levels of reactive oxygen species in human corneal epithelial cells. In human corneal epithelial cells, secretion of IL-6 and IL-8 in both apical and basolateral media was promoted significantly by perfluorooctanoic acid treatment. Stimulation with the basolateral media from perfluorooctanoic acid-treated human corneal epithelial cells induced inflammation in human corneal endothelial cells. The treatment of retinal pigment epithelial cells with the basolateral media from stimulated human corneal endothelial cells also elicited the secretion of proinflammatory cytokines. The results indicate that perfluorooctanoic acid exposure impaired the tight junction of corneal cells and caused inflammatory reactions in the retina. Exposure of the cornea to perfluorooctanoic acid contained in particulate matter might induce oxidative stress and inflammation in the retina and represent a risk factor for age-related macular degeneration.

Autophagy mediates perfluorooctanoic acid-induced lipid metabolism disorder and NLRP3 inflammasome activation in hepatocytes

Perfluorooctanoic acid (PFOA) has applications in numerous industrial products and is an industrial waste that is persistently present in the environment. Exposure to PFOA results in nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms remain unclear. In this study, male C57BL/6 mice were exposed to PFOA (1 mg/kg/day) for 4 weeks to evaluate the effect of PFOA, and the human liver cell line (L-02) was used to observe the direct effect of PFOA in vitro. After PFOA exposure, the expression of genes related to hepatic lipogenesis, the NLRP3 inflammasome, and autophagy were measured. We found that exposure to PFOA induced lipid accumulation and stimulated lipogenesis in both mouse livers and L-02 cells. In addition, increased NLRP3 aggregation and enhanced production of IL-1β occurred after PFOA treatment. We also found that PFOA exposure induced autophagosome formation and p62 accumulation, indicating blockage of autophagic flux. Rapamycin alleviated PFOA-induced lipid accumulation and NLRP3 inflammasome activation by activating autophagic flux. Conversely, chloroquine, an autophagic flux inhibitor, exacerbated PFOA-induced lipid accumulation and NLRP3 inflammasome activation. Collectively, these results provide evidence to show that PFOA-induced blockade of autophagic flux causes an increase in lipid synthesis and inflammation in vivo and in vitro.

Abstract A12: Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA)

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption, and pancreatitis, all of which share the ability to generate oxidative stress—a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre (KC) model. For our studies, KC mice received either tap water or tap water containing 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&amp;E stain), Alcian Blue (AB) staining, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Amylase, lipase, PFOA, and inflammatory cytokines were quantified in serum. Our results show that PFOA accumulates in the serum and pancreas of treated KC mice. In 6-month-old KC mice, exposure to PFOA increased the total AB+ lesion area from 4.87% ± 1.65% to 7.71% ± 3.01%. While the average lesion size was similar between control and PFOA-treated groups, the number of lesions per mm2 increased from 8.73 ± 3.19 to 16.73 ± 6.31, demonstrating that PFOA exposure leads to increased lesion development. qPCR analysis of pancreata verified increased expression of lesion markers CK19 and Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Collectively, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model, which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biologic persistence, suggest that it may participate in promoting pancreatic cancer progression. Citation Format: Barbara A. Hocevar, Lisa M. Kamendulis. Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA) [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A12.

Perfluorooctanoic acid activates the unfolded protein response in pancreatic acinar cells.

Perfluoroalkyl substances, such as perfluorooctanoic acid (PFOA), are widely used in consumer and industrial applications. Human epidemiologic and animal studies suggest that PFOA exposure elicits adverse effects on the pancreas; however, little is known about the biological effects of PFOA in this organ. In this study, we show that PFOA treatment of mouse pancreatic acinar cells results in endoplasmic reticulum (ER) stress and activation of the protein kinase-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase/endonuclease 1α (IRE1α), and activating transcription factor 6 arms of the unfolded protein response (UPR) pathway. PFOA-stimulated activation of the UPR was blocked by pretreatment with specific PERK and IRE1α inhibitors and the chemical chaperone 4-phenyl butyrate, but not the antioxidants N-acetyl- l-cysteine and Tiron. PFOA treatment led to increased cytosolic Ca+2 levels and induction of the UPR was blocked by an inhibitor of the inositol 1,4,5-trisphosphate receptor. These findings indicate that PFOA-induced ER stress may be the mechanistic trigger leading to oxidative stress in the pancreas.

Perfluorooctanoic Acid (in the Presence of Fetal Bovine Serum) Induces Proliferation in ERα Positive and ERα Negative Breast Cancer Cell Lines

Perfluorooctanoic acid (PFOA) is a synthetic chemical belonging to a larger group of fluorotelomers. These compounds have been used in the production of both industrial and consumer products as surfactants and due to their chemical nature are environmentally persistent pollutants. Individuals can be exposed to PFOA through ingesting PFOA‐contaminated water or food. PFOA has been shown to have a half‐life of approximately 4 years in humans. While the long‐term effects of PFOA are largely unknown, there is increasing evidence suggesting it to be an endocrine disruptor. Studies have shown that PFOA binds to and activates peroxisome‐proliferator‐activated receptor α (PPARα), which can regulate the expression of other genes and receptors such as the other PPAR isoforms as well as estrogen receptor α (ERα).Previous experiments in our lab demonstrated that PFOA treatment of MCF‐7 breast cancer cells (an ERα positive cell line) decreased expression of ERα mRNA and protein, and decreased cell viability by ~20% within 48h of treatment compared to DMSO controls. However, these cells were treated in the absence of fetal bovine serum (FBS). When we repeated these experiments without serum withdrawal, we initially noted a tendency towards increased proliferation in MCF‐7 cells treated with 50μM and 100μM PFOA at both 24h and 48h compared to control. To further examine the role of ERα in this PFOA‐induced proliferation, we carried out additional experiments in MCF‐7 cells along with experiments in another ERα‐positive cell line, T47D, as well as an ERα‐negative cell line, MDA‐MB‐231. There was a tendency for increased viability in all three cell lines when treated with PFOA, although only the MDA‐MB‐231 cells and the T47D cells showed a statistically significant (~20%) increase at 48h with 50μM PFOA and 100μM PFOA, respectively. These data suggest that the PFOA‐induced increase in cell viability in these cell lines is not dependent on ERα expression. In addition, the opposing effects of PFOA on proliferation in MCF‐7 cells in the presence and absence of FBS demonstrates the importance of accurately and completely reporting cell culture and treatment conditions.Support or Funding InformationAugusta University Department of Biological Sciences, Augusta University Center for Undergraduate Research and Scholarship

Gestational Perfluorooctanoic Acid Exposure Inhibits Placental Development by Dysregulation of Labyrinth Vessels and uNK Cells and Apoptosis in Mice.

Perfluorooctanoic acid (PFOA) is a widely used perfluorinated compound and known to cause developmental toxicity which includes the increase of resorbed embryo, decrease of fetal survival, and fetal growth retardation. Nevertheless, whether it is associated with alteration of placental development remains unknown. Pregnant mice were gavaged with 0, 2.5, 5, 10 mg PFOA /kg/day from pregnancy day (PD) 1 to PD 13. Results showed that PFOA exposure markedly decreased the placental weight and caused interstitial edema of placenta. Laminin staining indicated that blood sinusoids area was shrunken in placenta of PFOA-exposed mice. Furthermore, PFOA treatment significantly reduced numbers of uNK cells. Western blot analysis revealed that levels of Bax and cleaved-caspase 3 proteins were markedly up-regulated in PFOA-treated groups. In addition, TEM examination showed that PFOA treatment caused rupture of nuclear membrane and nuclear pyknosis and fragmentation. Thus, our results suggested that gestational PFOA exposure significantly inhibited development of early placenta through shrinkage of labyrinth vessels and downregulation of uNK cells and apoptosis induction, which may result in adverse gestational outcomes.

Fetal bovine serum attenuating perfluorooctanoic acid-inducing toxicity to multiple human cell lines via albumin binding

Perfluorooctanoic acid (PFOA), as a typical emerging organic pollutant, can interact with serum albumin. However, it remains to characterize the binding of PFOA with serum albumin and to address the role of this interaction in related toxic effects. We aimed to characterize the interaction between PFOA and albumin for understanding the effects of this interaction on the uptake, distribution, and toxicity of PFOA in human cells. The results showed that viable cell count was significantly enhanced by addition of fetabl bovine serum (FBS) into cell culture medium with 300 μM PFOA treatment. PFOA mainly existed as complexed with FBS, at FBS concentration > 10%, which substantially reduced the absorption efficiency of all cell lines to PFOA. The majority of PFOA was accumulated in the cytosolic fraction, followed by nuclei, and mitochondria. Conclusively, our study suggests that the complexation of organic contaminants with proteins might mitigate their toxicity by reducing cellular uptake.

Metabolomic analysis reveals metabolic alterations of human peripheral blood lymphocytes by perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) is a dispersive persistent organic pollutant in the environment. Accumulating reports suggest that PFOA is toxic to human lymphocytes; however, the toxicological effects of PFOA on these cells remain largely unclear. In this study, ultra-performance liquid chromatography (UPLC)-based metabolomic analysis was employed to identify metabolites in human peripheral blood lymphocytes and to assess the metabolic alterations caused by PFOA exposure. Our comparative metabolomic analysis results demonstrated that PFOA treatment could increase the level of organic acids and reduce the level of lipid molecules. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation further highlighted the fact that the PFOA treatment interfered with the metabolism of amino acids, carbohydrates and lipids, which may lead to disruption of the immune system.

Developmental exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) selectively decreases brain dopamine levels in Northern leopard frogs

Per- and polyfluoroalkyl substances (PFAS) are synthetic compounds that are a major public health concern due to widespread use, long environmental and biological half-lives, detection in most human plasma samples, and links to multiple adverse health outcomes. The literature suggests that some PFAS may be neurotoxic. However, there are major gaps in the literature with respect to how environmentally-relevant doses during development may influence the nervous system. To address this gap, we utilized a sentinel species, Northern leopard frogs (Lithobates pipiens) to determine the effects of developmental exposure to environmentally relevant perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on major neurotransmitter systems. Frog larvae at Gosner stage 25 were exposed to 10, 100, or 1000 ppb PFOS or PFOA for 30 days before neurochemical analysis. High performance liquid chromatography (HPLC) with electrochemical detection or fluorescent detection assays was used to measure neurotransmitter levels, which were normalized to protein levels in each sample. Dopamine (DA) decreased significantly in the brains of frogs treated with PFOA (1000 ppb) and PFOS (100 and 1000 ppb). Significant increases in DA turnover also resulted from PFOA and PFOS treatment. Neither PFOS, nor PFOA produced detectable alterations in serotonin (nor its metabolite), norepinephrine, gamma-amino butyric acid (GABA), glutamate, or acetylcholine. PFAS body burdens showed that PFOS accumulated relative to dose, while PFOA did not. These data suggest that DArgic neurotransmission is selectively affected in developmentally exposed amphibians and that PFAS should be evaluated for a potential role in diseases that target the DA system.

Maternal exposure to perfluorooctanoic acid (PFOA) causes liver toxicity through PPAR-α pathway and lowered histone acetylation in female offspring mice.

The study was conducted to investigate the liver toxicity in female offspring mice induced by maternal exposure to perfluorooctanoic acid (PFOA). Fifty pregnant Kunming mice were randomly divided into 5 groups with 10 of each, which were treated with 0.2 mL PFOA solution dissolved with deionized water at 0, 1, 2.5, 5, and 10 mg/kg BW, respectively, from the pregnancy day (PND) 0 to day 17. Female offspring mice were sacrificed to collect serum and liver at postpartum day 21. The results showed that PFOA significantly reduced the body weight at weaning and the survival rate of the female offspring mice (P < 0.01) increased the liver index of the pups (P < 0.01). Meanwhile, PFOA also caused hepatic bleeding, local necrosis, and enlargement of hepatocytes and vacuolization. The levels of serum AST, ALT, SOD, and CAT in PFOA treatment group were upregulated significantly (P < 0.01). The expressions of Acot1, Acox1, and Acsl1 genes were increased significantly (P < 0.01). The expression of PPAR-α gene was decreased significantly (P < 0.01). There was no significant difference in the expression of Cpt1a gene among the 5 groups. HAT activity was reduced significantly and HDAC activity was increased significantly. The expression of anti-acetyl-histone H3 and acetyl-histone H4 was reduced significantly. Thus, our findings indicate that exposure to PFOA during pregnancy affects the growth and development of the pups and causes liver damage, disrupting the secretion of enzymes involved in fatty acid oxidation induced by PPAR-α, leading to liver oxidative stress and a decrease in the degree of histone acetylation. Elevated HDAC may aggravate downstream fatty acid metabolism disorders through PPAR-α.

Comparative Hepatotoxicity of Novel PFOA Alternatives (Perfluoropolyether Carboxylic Acids) on Male Mice.

As novel alternatives to perfluorooctanoic acid (PFOA), perfluoropolyether carboxylic acids (multiether PFECAs, CF3(OCF2) nCOO-, n = 2-4) have been detected in various environmental matrices; however, public information regarding their toxicities remains unavailable. To compare the hepatotoxicity of multiether PFECAs (e.g., PFO2HxA, PFO3OA, and PFO4DA) with PFOA, male mice were exposed to 0.4, 2, or 10 mg/kg/d of each chemical for 28 d, respectively. Results demonstrated that PFO2HxA and PFO3OA exposure did not induce marked increases in relative liver weight; whereas 2 and 10 mg/kg/d of PFO4DA significantly increased relative liver weight. Furthermore, PFO2HxA and PFO3OA demonstrated almost no accumulation in the liver or serum; whereas PFO4DA was accumulated but with weaker potential than PFOA. Exposure to 10 mg/kg/d of PFO4DA led to 198 differentially expressed liver genes (56 down-regulated, 142 up-regulated), with bioinformatics analysis highlighting the urea cycle disorder. Like PFOA, 10 mg/kg/d of PFO4DA decreased the urea cycle-related enzyme protein levels (e.g., carbamoyl phosphate synthetase 1) and serum ammonia content in a dose-dependent manner. Both PFOA and PFO4DA treatment (highest concentration) caused a decrease in glutamate content and increase in both glutamine synthetase activity and aquaporin protein levels in the brain. Thus, we concluded that PFO4DA caused hepatotoxicity, as indicated by hepatomegaly and karyolysis, though to a lesser degree than PFOA, and induced urea cycle disorder, which may contribute to the observed toxic effects.

Perfluorooctanoic acid exposure induces apoptosis in SMMC-7721 hepatocellular cancer cells

To better understand the toxicological activities of perfluorooctanoic acid (PFOA), we examined the effects of PFOA on apoptosis and its molecular mechanism in SMMC-7721 hepatoma cells. Cell viability was evaluated by MTT assay and apoptosis was determined by flow cytometry. Western blot and quantitative real-time PCR were used to examine the protein and gene expressions of Bax and Bcl-2. Our results showed that PFOA inhibited SMMC-7721 cell growth and induced apoptosis. PFOA treatment increased Bax expression and increased Bcl-2 expression at both gene and protein levels. Our study demonstrated that PFOA had toxic effects on SMMC-7721 cells, such as inhibiting cell proliferation and inducing apoptosis. Furthermore, we showed that PFOA-mediated induction of apoptosis involved inducing Bax and decreasing Bcl-2 expression as a molecular mechanism of its toxicological effects.

Induction of hepatic miR-34a by perfluorooctanoic acid regulates metabolism-related genes in mice

Perfluorooctanoic acid (PFOA) is a widespread organic pollutant with various toxicological impacts on the liver. Members of the miR-34 family are P53-targeted growth suppressors. We found that PFOA exposure (5 mg/kg/d PFOA for 28 d) resulted in a significant increase of miR-34a in the livers of mice but had no effect on either miR-34b or miR-34c. We knocked out miR-34a in mice to explore the role of elevated miR-34a in PFOA-induced liver toxicity. Compared with the corresponding untreated control, significant increases in liver weight as well as serum alanine transaminase, aspartate aminotransferase, and cholinesterase levels were observed in miR-34a−/− and wild-type mice after PFOA exposure. Hepatic cells showed similar swelling in both miR-34a−/− and wild-type mice after PFOA treatment. Hepatic RNA-sequencing (RNA-seq) showed that PFOA led to significant alteration in lipid metabolism genes, especially those involved in the peroxisome proliferator-activated receptor pathway, in both wild-type and miR-34a null mice. With or without PFOA treatment, relatively fewer genes were altered in miR-34a−/− livers compared to wild-type livers. Among the changed genes by miR-34a, the most dominant were metabolism-related genes, such as Fabp3, Cyp7a1, and Apoa4. Our in vivo study indicated that miR-34a mainly exerts a metabolic regulation role, rather than the pro-apoptosis and cell cycle arrest role reported previously by many in vitro studies. In addition, although hepatic P53 was unchanged, the active type of P53 (acetylated P53 (acetyl-p53, Lys379)) was markedly altered under PFOA treatment. Therefore, the increase in acetylated P53 may have activated the transcription of miR-34a in mouse livers after PFOA treatment.

Lipid accumulation responses in the liver of Rana nigromaculata induced by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a perfluorinated compound that is widely distributed, is persistent in the environment, and has a low-level chronic exposure effect on human health. The aim of this study was to investigate the peroxisome proliferator activated receptors γ (PPARγ) and the sterol regulatory element-binding protein 2 (SREBP2) signaling pathways in regulating the lipid damage response to PFOA in the livers of amphibians. Male and female frogs (Rana nigromaculata) were exposed to 0, 0.01, 0.1, 0.5 and 1 mg/L PFOA. After treatment, we evaluated the pathological changes in the liver by Oil Red O, staining and examined the total cholesterol (T-CHO) and triglyceride (TG) contents. The mRNA expression levels of PPARγ, Fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), Glycerol-3-phosphate acyltransferase (GPAT), SREBP2 and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The administration of PFOA caused marked lipid accumulation damage in the amphibian livers. The T-CHO contents were elevated significantly after PFOA treatment; these results show a dose-dependent manner in both sexes. The TG content showed a significant increase in male livers, while it was elevated significantly in female livers. The RT-PCR results showed that the mRNA expression levels of PPARγ, ACC, FAS, GPAT, SREBP2 and HMG-CoA were significantly dose-dependently increased in the PFOA-treated groups compared with those of the control group. Our results demonstrated that PFOA-induced lipid accumulation also affected the expression levels of genes FAS, ACC, GPAT and HMG-CoA in the PPARγ and SREBP2 signaling pathways in the liver. These finding will provide a scientific theoretical basis for the protection of Rana nigromaculata against PFOA effects.

Abstract 3095: Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA)

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption and pancreatitis; all of which share the ability to generate oxidative stress - a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~ 4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre model. For our studies, LSL-KRasG12D;Pdx-1Cre mice received either tap water or tap water supplemented with 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&amp;E stain), immunohistochemistry for CK19, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Serum was utilized for quantitation of amylase, lipase, PFOA and inflammatory cytokine levels. Our preliminary results show that PFOA accumulates in the serum and pancreas of treated LSL-KRasG12D;Pdx-1 Cre mice. In addition, exposure to PFOA increased the CK19+ lesion area in LSL-KRasG12D;Pdx-1 Cre mice indicating an acceleration of pancreatic cancer progression. qPCR analysis of pancreata verified increased expression of both CK19 and the early lesion marker Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Together, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biological persistence, suggest that it could be a prominent agent promoting pancreatic cancer progession. Citation Format: Barbara A. Hocevar, Lisa M. Kamendulis. Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3095.

Perfluorooctanoic acid stimulates ovarian cancer cell migration, invasion via ERK/NF-κB/MMP-2/-9 pathway

As widely used in consumer products, perfluorooctanoic acid (PFOA) has become a common environmental pollutant, which has been detected in human serum and associated with cancers. Our previous study showed that PFOA is a carcinogen that promotes endometrial cancer cell migration and invasion through activation of ERK/mTOR signaling. Here, we showed that PFOA (≥100 nM) treatment also stimulated A2780 ovarian cancer cell invasion and migration, which correlated with increased matrix metalloproteinases MMP-2/-9 expression, important proteases associated with tumor invasion and migration. Notably, PFOA treatment induced activation of ERK1/2/ NF-κB signaling. Pre-treatment with U0126, an ERK1/2inhibitor；or JSH-23, a NF-kB inhibitor, can reverse the PFOA-induced cell migration and invasion. Consistent with these results, inhibiting ERK1/2 or NF-κB signaling abolished PFOA-induced up-regulation of MMP-2/-9 expression. These results indicate that PFOA can stimulate ovarian cancer cell migration, invasion and MMP-2/-9 expression by up-regulating ERK/NF-κB pathway.