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. 2025 Oct 15;10(42):50285–50297. doi: 10.1021/acsomega.5c07073

Perfluoroundecanoic Acid Inhibits Liver Cell Development in Pubertal Male Rats via Inducing Oxidative Stress

Zengqiang Li †,‡, Chaoqun Guo §, Xinyi Ye †,‡, Qiang Xu ∥, Lijun Weng †,‡, Youguang Gao †,‡,*, Ren-shan Ge ∥,*, Xianzhong Lin †,‡,*
PMCID: PMC12573167  PMID: 41179154

Abstract

Perfluoroundecanoic acid (PFUnA) is a long-chain perfluoroalkyl substance. However, whether PFUnA affects liver development during puberty is still unclear. Male 35-day-old Sprague–Dawley rats were gavaged with 0 (corn oil), 1, 5, and 10 mg/kg/day of PFUnA for 21 days. Liver weight was remarkably increased by PFUnA at ≥1 mg/kg, while serum alanine aminotransferase levels were higher at 5 and 10 mg/kg. PFUnA upregulated the expression of Lxrα, Hmgcr, Srebp2, and Cyp7a1 and downregulated the expression of Abcg8, Ldlr, Sod1, and Cat. These changes were also confirmed at the protein level. It increased the release of inflammatory factors (IL-1β, IL-6, and TNF-α). It also downregulated the fibrosis-related gene expression of Adamts6 and Col4a1. Similar results were obtained in HepG2 cells treated with 50 μM PFUnA in vitro. PFUnA induced oxidative stress and perturbed lipid metabolism. Overall, PFUnA may induce oxidative stress and disrupt lipid metabolism to have an inhibitory influence on pubertal liver development.

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1. Introduction

Globally, nonalcoholic fatty liver disease (NAFLD) ranks among the most widespread liver disorders. Scholars have lately proposed renaming it as metabolic dysfunction-associated steatotic liver disease (MASLD). According to recent epidemiological data, the incidence of NAFLD has significantly increased from a baseline of 25.24% in 2016 to 32.4% in 2022, exceeding an earlier estimate. Accelerated research and development efforts are desperately needed to find safe and efficient prevention strategies for NAFLD, given the skyrocketing incidence of NAFLD.

A complicated range of clinical disorders is included in fatty liver disease, which starts with steatosis or the buildup of lipids in liver cells. As the illness worsens, steatohepatitis, a more severe condition marked by necrotizing inflammation and quicker fibrosis, may develop, ultimately culminating in the irreversible condition of cirrhosis, and even hepatocellular carcinoma (HCC). Numerous factors can contribute to the onset and progression of NAFLD. Obesity, insulin resistance, and metabolic syndrome are strongly associated with an increased risk of NAFLD, which is impacted by intricate interactions between genetic and environmental factors. NAFLD and its possible consequences have a complex pathophysiology, with multiple variables that may work alone or in combination to promote the development of nonalcoholic steatohepatitis (NASH), a process that is still poorly understood.

Human health is at risk due to exposure to a variety of synthetic organic compounds known as poly- and perfluoroalkyl substances (PFAS). PFAS, which encompass poly- and perfluoroalkyl carboxylic acids and poly- and perfluoroalkyl sulfonic acids with different carbon chain lengths (C4–C14), consist of more than 9,000 unique chemical compounds. Because the hydrogen atoms in the carbon chains of poly- and perfluoroalkyl sulfonic acids are partially or entirely replaced by fluorine atoms, these compounds are extremely persistent in the environment. PFAS find their way into water, the atmosphere, sludge, and other environmental media due to their extensive use, where they bioaccumulate through the food chain. Due to the adverse effects of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA), which are currently prohibited in many regions, manufacturers have turned to substituting alternative long-chained PFAS (>C8), like perfluoroundecanoic acid (PFUnA, Figure A), for PFOS and PFOA. Human exposure to PFUnA occurs via both direct and indirect routes. As a result of greater application, the amount of PFUnA has lately grown. In Hokkaido, Japan, the mean PFUnA level in pregnant women’s serum was 1.5 ng/mL. The elimination half-life of PFUnA in humans is 9.7 years, which is far longer than that of PFOA (3.8 years). This implies that PFUnA persists in the human body longer than PFOA. Serum samples from people in Siheung, South Korea, showed PFUnA concentrations ranging from 1.11 to 4.58 ng/mL, whereas the median PFUnA concentration found in breast milk from people living close to Seattle, Washington, in the United States, was 4.43 pg/mL.

1.

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PFUnA chemical structure, regimen, and serum enzyme levels following in vivo PFUnA administration. (A) The PFUnA chemical structure; (B) regimen, rats were given PFUnA by gavage for 21 days at doses of 0, 1, 5, and 10 mg/kg/day; (C) serum alanine aminotransferase (ALT); (D) aspartate aminotransferase (AST); (E) γ-glutamyl transpeptidase (γ-GT); (F) total protein (TP); (G) albumin (ALB); (H) albumin-to-globulin ratio (A/G); (I) triglycerides (TG); (J) total cholesterol (TC). Values represent mean ± SEM (n = 6). Significant differences compared to the control (0 mg/kg) are indicated by *p < 0.05 and **p < 0.01.

Animal studies have shown that exposure of rodents to PFAS causes a series of manifestations of liver function damage such as hepatic steatosis, elevated liver enzymes, hepatocyte hypertrophy, and liver enlargement. Alternatively, PFAS exposure may promote hepatic steatosis by disrupting hepatic lipid homeostasis. However, there is little literature on the effects of PFUnA on liver development. During puberty, various organs undergo rapid developmental changes and become vulnerable targets for PFAS. Their effects on pubertal development are of significant concern. According to epidemiological studies, PFUnA may potentially affect growth and development, cardiovascular health, and the immune system. While there is little research to date carried out on the probable carcinogenic and genotoxic effects of PFUnA in animal models, in the limited animal studies of PFUnA, exposure to PFUnA has been linked to low birth weight, delayed puberty, decreased breastfeeding, altered reproductive cycles, immune system issues, cancer, and changes in liver size, among others. , As far as we are knowledgeable, only a limited number of studies have assessed the toxicity of PFUnA in animals. As a result, there is a dearth of information on cellular and systemic toxicity, as well as genotoxicity, that has not been thoroughly explored.

A potential mechanism by which PFUnA acts in both humans and animals is by producing oxidative stress. Many environmental pollutants have the ability to promote oxidative stress, which raises reactive oxygen species (ROS) levels, damages DNA, and causes lipid peroxidation; PFUnA might induce similar mechanisms.

2. Materials and Methods

2.1. Materials and Animals

Key reagents (Table S1), qPCR primers (Table S2), and antibodies (Table S3) are listed in the Supporting Information. This study used 28-day-old male Sprague–Dawley rats (Shanghai Laboratory Animal Center) under a protocol approved by the Animal Care and Use Committee of Fujian Medical University, adhering to all ethical guidelines for laboratory animal care and use.

2.2. Animal Experiment

All experimental animals in new environment (temperature, 23 °C; relative humidity, 45–55%; and photoperiod, 12 h light/12 h dark) were fed on a regular diet for 1 week. From lowest to highest, the rats’ weights were numbered. After that, they were evenly split into four groups (n = 6 per group) according to their weights, making sure that there were no notable variations between the groups. The animals in the different groups were fed 0, 1, 5, and 10 mg/kg PFUnA. The designated group administered PFUnA to rats after it was dissolved in corn oil. The oral route was chosen based on the likelihood of human PFUnA exposure via the food chain. Based on the findings of our previously published PFUnA toxicological study, which showed that PFUnA inhibits testosterone at a dose of 1 mg/kg during puberty, the dosage was chosen. Following 21 days of gavage, rats underwent euthanasia by carbon dioxide asphyxiation on postnatal day (PND) 56. Liver and blood samples were obtained for additional examination.

2.3. Biochemical Analysis

We utilized a Chemray 800 automatic blood biochemical analyzer (Rayto Life and Analytical Sciences Co., Ltd, Shenzhen, China) to assess the levels of serum triglycerides (TG), total cholesterol (TC), total protein (TP), albumin (ALB), γ-glutamyl transpeptidase (γ-GT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Commercial kits from Jianglai Industrial (Shanghai, China) were used to measure the liver’s TC and TG levels.

2.4. Histological Analysis

Dehydrated rat liver tissues were embedded. Following the cutting of cross sections (6 μm), the sections were histochemically stained using hematoxylin–eosin staining solution. Images were captured at 10× and 40× magnification using a Nikon Eclipse E100 microscope (Nikon, Japan). CaseViewer 2.4.0 was used to view and examine the tissue slices.

2.5. qPCR

qPCR analysis was conducted as described. qPCR was performed with a SYBR Green kit and Table S2. Gene expression was quantified using the standard curve method and normalized to Gapdh.

2.6. Western Blotting

Western blotting and sample preparation were carried out as described before. The following antigens were detected: here are the full names of the requested genes and proteins along with their primary functions: PPAR (Peroxisome Proliferator Activated Receptor ), SREBP2 (Sterol Regulatory Element-Binding Protein 2), LXRα (Liver X Receptor Alpha (NR1H3), LDLR (Low-Density Lipoprotein Receptor), HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase), ABCG8 (ATP-Binding Cassette Subfamily G Member 8), CYP7A1 (Cholesterol 7-Alpha Hydroxylase), SOD1 (Superoxide Dismutase 1), CAT (Catalase), COL4A1 (Collagen Type IV Alpha 1 Chain), ADAMTS6 (ADAM Metallopeptidase with Thrombospondin Type 1 Motif 6), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase). Using Image Lab software, the protein’s intensity was measured and calibrated to the housekeeping protein GAPDH.

2.7. Malondialdehyde (MDA) Measurement

Tri-unsaturated fatty acid hydroperoxides break down to produce malondialdehyde (MDA), a chemical molecule that is a type of ROS. MDA was detected according to the kit instructions, as previously described.

2.8. Immunohistochemistry

Immunohistochemistry was conducted as previously described. Six livers per group, with random sections selected for embedding, were used. Five nonserial sections (6 μm) were analyzed per sample. The immunohistochemical staining procedure started with heating to display the antigen and then using 0.5% H2O2 to block endogenous peroxidase. Incubation of the sections was performed sequentially with primary and secondary antibodies.

2.9. Liver Cytokine Determination

With the aid of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) platinum enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Inc., San Diego, CA, USA), the levels of liver IL-1β, IL-6, and TNF-α were measured. The manufacturer’s instructions were followed for conducting the tests.

2.10. RNA Sequencing, Data Analysis, and Biological Pathway Analysis

Four livers from each group, 0 mg/kg (Group A) and 10 mg/kg (Group D) PFUnA-treated rats, were randomly picked for RNA isolation. Because PFUnA had a stronger effect on the levels of hepatic cytokines, Group D was selected. The RNA-seq library was made as previously described after total RNAs were extracted from the livers and enriched using oligo-dT magnetic beads. The Illumina NovaSeq 6000 device was used to sequence the RNA-seq libraries. After analyzing the data, we looked for genes and transcripts that were differently expressed using the R package. The following statistical calculations and plots were performed: scatter plot, volcano plot, Gene Ontology (GO) analysis, route analysis, and hierarchical clustering.

2.11. Experiments Using HepG2

The human hepatocellular carcinoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, VA) and maintained in high-glucose DMEM supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin. The cells were then incubated at 37 °C in 5% CO2. Following the manufacturer’s instructions, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was used to assess the impact of PFUnA on cell viability (Nacalai Tesque).

2.12. Statistical Analysis

Mean ± standard error (SEM) is used to display all data. Statistical analyses were performed on data from individual litters (dams; n = 6/group) after verifying the assumptions of normality and homogeneity of variance. GraphPad Prism was used for statistical analysis. The following were deemed statistically significant: p < 0.05, 0.01, or 0.001.

3. Results

3.1. General Parameters of Toxicology

Prior to the trial, the body weights of the pubertal rats were distributed uniformly, as shown in Table . Rats were given PFUnA intragastrically from PND 35 to PND 56 (Figure B). At dosages of 5 and 10 mg/kg, PFUnA dramatically decreased body weight after 21 days of therapy (Table ). This is consistent with a previous study. However, Table indicates that both the liver weight and the relative liver weight increased significantly at doses of 5 and 10 mg/kg PFUnA. Even though the PFUnA group’s liver weight increased by 1 mg/kg, there was no discernible change in their liver index when compared to the control group. Throughout the study, no mortality or morbidity was observed in any group of rats.

1. General Reproductive Parameters in Rats Exposed to Perfluoroundecanoic Acid (PFUnA) .

  PFUnA (mg/kg/day)
Parameter 0 1 5 10
Number 6 6 6 6
Body weight (g)  
Before treatment 154.7 ± 3.16 158.9 ± 1.53 153.7 ± 3.10 156.2 ± 2.62
After treatment 295.5 ± 3.66 301.4 ± 4.74 261.5 ± 7.99, 174.8 ± 9.16
Liver weight (g) 10.25 ± 0.25 11.27 ± 0.25 12.70 ± 0.37 13.65 ± 0.38
Liver/body weight (%) 3.47 ± 0.11 3.74 ± 0.05 4.98 ± 0.09, 8.74 ± 0.81
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a

p < 0.05 vs control (0 mg/kg).

b

p < 0.05 vs 5 mg/kg group.

c

Data represent mean ± SEM (n = 6).

3.2. PFUnA Affects Liver Function and Serum Lipid Levels

At a dose of 5 mg/kg, PFUnA markedly increased serum ALT levels (Figure C). It significantly increased serum ALT, AST, and γ-GT levels at 10 mg/kg (Figure C–E). PFUnA significantly reduced serum TP, ALB, and A/G levels at 10 mg/kg (Figure F–H). At whatever test dose was used, it had no effect on serum TG levels (Figure I). However, PFUnA significantly increased serum TC levels at doses of 5 and 10 mg/kg (Figure J). Collectively, our data indicate that PFUnA can alter hepatic lipid metabolism and impair liver function.

3.3. PFUnA Induces Liver Lipid Level Increases

PFUnA significantly increased liver TC levels at a dose of 10 mg/kg (Figure A). At whatever test dose, it had no effect on liver TG levels (Figure B). Liver hypertrophy was observed after PFUnA exposure during puberty (Figure S1).

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Liver histochemical staining and immunohistochemistry of LXRα and LDLR in rat liver sections and lipid levels of rats after PFUnA treatment in vivo. (A) Total cholesterol level; (B) TG level. (C,D) Representative histological images of liver sections stained with H&E staining. Scale bar: 50 μm with 10× magnification; (E) LXRα staining; (F) LDLR staining. The black arrow points to the punctate necrosis of hepatocytes. The black arrow points to the punctate necrosis of hepatocytes; the red arrow points to the vasculitis. Bar = 50 μm. Values are mean ± SEM, n = 6. *p < 0.05 vs control (0 mg/kg). Red arrow: vascular inflammation; black arrowhead: apoptosis.

3.4. PFUnA Induces Inflammatory Infiltration of Liver Tissue

Histological staining with H&E revealed that PFUnA was associated with the occurrence of vascular inflammation (Figure C,D). The 10 mg/kg group stage was characterized by mixed hepatocyte ballooning (grade 1), inflammation (grade 3), fibrosis (grade 1), and the absence of steatosis (Figure C,D), with an NAFLD Activity Score (NAS) of 4 (Table S4).

One important factor influencing the amount of circulating plasma lipoproteins is the LDLR (low-density lipoprotein receptor). It can induce the absorption of serum cholesterol by hepatocytes. LXRα is mostly present in cells and tissues that are involved in metabolism, including adipose tissue and the liver. In the 5 and 10 mg/kg groups, we observed that PFUnA enhanced the area of LXRα-positive cells (Figure E). This indicates that PFUnA induces increased LXRα expression in liver cells. However, PFUnA decreased the area of LDLR-positive cells at doses of 5 and 10 mg/kg (Figure F). This suggests that PFUnA suppressed the expression of LDLR in liver cells.

3.5. PFUnA Affects Gene Expression in Liver Cells

PFUnA significantly upregulated the expression of Lxrα, and downregulated the expression of Ldlr at a dose of 5 mg/kg. It significantly upregulated the expression of Lxrα, Srebp2, Hmgcr, Pparα, and Cyp7a1 in the 10 mg/kg group (Figure ). But at 10 mg/kg, it significantly decreased the expression of Ldlr and Abcg8 (Figure ). This indicates that PFUnA regulates the expression of lipid-metabolism-related genes. Furthermore, we discovered that PFUnA (10 mg/kg) markedly upregulated the expression of fibrosis-related genes (Adamts6 and Col4a1).

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On day 56 following PFUnA treatment in vivo, gene expression was detected in the liver. Lipid-metabolism-related genes: Lxrα, Abcg5, Abcg8, Abca1, Pcsk9, Ldlr, Srebp2, Hmgcr, Cyp7a1, Soat, Pparα, ApoC3, and ApoC4; fibrosis-related genes: Col4a1 and Adamts6. The mRNA levels were adjusted to Gapdh. Values are mean ± SEM (n = 6). Significant differences compared to the control (0 mg/kg) are indicated as *p < 0.05 and **p < 0.01.

3.6. PFUnA Affects the Protein Levels of Liver Cells

PFUnA significantly increased the protein levels of LXRα, HMGCR, SREBP2, PPAR, and CYP7A1, in parallel with their mRNA levels (Figure ). It significantly decreased the protein levels of LDLR and ABCG8, in parallel with their mRNA levels (Figure ).

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Liver protein levels on day 56 following PFUnA treatment in vivo. (A) Western blot image; (B–H) HMGCR, PPAR, SREBP2, LXRα, LDLR, ABCG8, and CYP7A1 levels. GAPDH-normalized protein levels (mean ± SEM, n = 5). *p < 0.05 vs control (0 mg/kg).

3.7. PFUnA Affects Lipid Peroxidation and Suppression of Antioxidants

The qPCR was used to measure antioxidant-related gene mRNAs (Sod1, Sod2, Gpx1, and Cat). PFUnA significantly upregulated the expression of Sod1 at a dose of 1 mg/kg and downregulated the expression of Cat at a dose of 5 mg/kg. A significant downregulation of Sod1 and Cat expression was observed at the 10 mg/kg dose (Figure A). Western blot analysis was performed to assess the expression of antioxidant-related proteins. Parallel to their mRNA levels, PFUnA markedly reduced their protein levels (Figure C). In order to investigate the effects of PFUnA, we also examined the MDA levels. At a dose of 10 mg/kg, PFUnA markedly raised MDA levels (Figure C). These findings suggest that PFUnA lowers endogenous antioxidant levels and induces lipid peroxidation.

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Gene expression and protein levels in the liver and malondialdehyde (MDA) following PFUnA treatment in vivo. (A) Gene expression (Sod1, Gpx1, Cat, Il-1β, Il-6, and Tnf-α); (B) inflammatory factor levels; (C) Western blot image and protein levels and MDA level. GAPDH-normalized protein levels (mean ± SEM, n = 5). *p < 0.05 vs control (0 mg/kg).

3.8. PFUnA Affects Inflammatory Cytokines and Fibrosis

The qPCR was used to measure inflammatory-related gene mRNAs (Il-1β, Il-6 and Tnf-α). PFUnA significantly upregulated the expression of Il-1β, Il-6, and Tnf-α at a dose of 10 mg/kg (Figure A). PFUnA significantly increased the tissue inflammatory cytokine levels of IL-1, IL-6, and TNF-α at a dose of 10 mg/kg, in parallel with their mRNA levels (Figure B). The 10 mg/kg dose caused an elevation in the levels of COL4A1 and ADAMTS proteins, which mirrored their transcriptional changes (Figure C). These indicate that PFUnA causes cellular inflammation and promotes liver fibrosis.

3.9. Pathway Analysis after the Treatment of PFUnA

To determine the mechanism of inhibition of lipid metabolism by PFUnA, we performed RNA-seq on PFUnA-treated rats. 18,260 transcripts were identified in the livers of two groups (0 and 10 mg/kg PFUnA). Figure B shows that 1316 of these transcripts were considerably downregulated (p < 0.05) and 1271 were significantly upregulated (p < 0.05). A heatmap analysis indicated a difference between the Control group and the PFUnA group (Figure A).

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In vivo PFUnA treatment alters the hepatic transcriptome. 0 mg/kg (Group A) and 10 mg/kg (Group D) PFUnA-treated rats were compared (n = 4). Heatmap showing the 2-fold difference in upregulated (red) and downregulated (blue) genes between Group D and Group A (A); scatter plot showing the 2-fold differences in up- and downregulated (red) genes between Groups D and A (B); for the classification of Biological Process (BP), Cellular Component (CC), or Molecular Function (MF), GO analysis was performed to identify upregulated genes in Group D as opposed to Group A (C).

GO analysis revealed that most downregulated genes were related to the immune system, cellular enzyme activity, lipid transport, and lipoprotein microparticles (Figure C), and most upregulated genes were related to lipid compound metabolism, steroid metabolism, the basolateral plasma membrane, cellular protein modifications, and preribosome (Figure C). Further pathway analysis revealed that the expression of 2 genes (Soat and Cyp7a1) in the cholesterol biosynthetic signaling pathway was upregulated by ≥2-fold (Figure ), and the expression of 5 genes (Apoc3, Apoa4, Abcg8, Abcg5, and Ldlr) was downregulated by ≥2-fold (Figure ). To validate the RNA-seq data, we performed qPCR (as shown in Figure ).

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Cholesterol metabolism and transport pathway of the liver after PFUnA treatment in vivo. Upregulated (red) and downregulated (blue) genes in Groups D and A are compared.

3.10. Effect of PFUnA on Gene Expression of HepG2 Cells In Vitro

Next, we used human HepG2 cells to examine the direct effects of PFUnA on hepatocytes. HepG2 cells were treated with PFUnA for 24 h after the viability of cells treated with less than 50 μM PFUnA was confirmed (Figure ). To explore whether PFUnA directly affects HepG2 cells’ lipid synthesis and metabolism, we examined the gene expression of HepG2 cells. PFUnA significantly upregulated Lxrα, Cyp7a1, Hmgcr, and Srebp2 at 50 μM. It significantly downregulated Abcg8, Ldlr, Sod1, and Cat, which is similar to the effects of PFUnA exposure in vivo (Figure ).

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Gene expression and cell viability in HepG2 cells after PFUnA treatment in vitro. Regimen, cell viability, and gene expression of Lxrα, Abcg8, Ldlr, Srebp2, Hmgcr, Cyp7a1, Sod1, and Cat. Data represent mean ± SEM (n = 4 independent isolations) of Gapdh-normalized mRNA levels. *p < 0.05 vs control (0 μM).

4. Discussion

This study demonstrated that by controlling inflammation and inhibiting lipid metabolism, PFUnA impaired hepatic development during puberty. In this work, we examined the impact of varying doses of PFUnA consumption on rat metabolism. Rats given dosages of 5 and 10 mg/kg of PFUnA showed reduced body weight, serum lipid TC levels, and liver hypertrophy following PFUnA exposure during adolescence compared to the control group. However, at doses of 10 mg/kg, the liver’s TC concentration increased. Despite the fact that the 1 mg/kg PFUnA dose had no effect on body weight, the liver weight increased noticeably. This suggests that PFUnA selectively targets liver tissue at low doses (1 mg/kg).

According to Hu et al., obesity was caused by fat macronutrients rather than proteins or carbohydrates. Weight loss was associated with lower blood lipids, which is in line with this study’s findings. However, rat liver weight, serum ALT levels, and total cholesterol deposition were all higher in the 10 mg/kg PFUnA consumption group than in the control group. Given that the PFUnA group’s body weight was much lower than that of the control group, we postulated that the rat’s cholesterol deposition was the source of the larger liver weights in the PFUnA group compared to the control group. Rats given 5 mg/kg of PFUnA did not exhibit any discernible cholesterol buildup in their livers, which is likely due to the rat’s dietary schedule. The 21-day exposure period may have been insufficient for steatosis development. Steatosis is encouraged by prolonged exposure to PFAS. Rats in the 10 mg/kg PFUnA group had cholesterol accumulation; however, this was not followed by severe hepatocyte lipidosis. The lack of an increase in hepatic TG levels could be the cause of this.

Nevertheless, it is unknown which PFUnA intake contributes to an increase in the TC concentration in rat livers. To elucidate the underlying mechanism, we profiled the mRNA expression of genes regulating fatty acid and cholesterol metabolism in rat liver. Endogenous synthesis, external absorption, efflux, and esterification are the four primary components of cholesterol metabolism, according to Luo et al. First, as the liver is the main location for the manufacture of cholesterol, higher levels of Hmgcr mRNA expression result in higher levels of cholesterol being synthesized there. HMGCR is the enzyme that limits the rate of the endogenous production route. A crucial component of cholesterol production, Srebp2 upregulation promotes the expression of the Ldlr and Hmgcr genes. Since hepatocytes internalize serum LDL-C through LDLR, upregulation of the Ldlr gene’s expression may facilitate hepatocytes’ absorption of serum cholesterol. In this study, rats given 10 mg/kg of PFUnA had levels of Srebp2 and Hmgcr mRNA expression that were higher than those of the control group. The increased cholesterol content in the rat livers at doses of 10 mg/kg PFUnA group may be due in part to the hepatic cholesterol synthesis induced by PFUnA ingestion through the SREBP2–HMGCR signaling pathway. However, we also found that although PFUnA upregulated the expression of Srebp2 but downregulated the expression of Ldlr, which suggested that Ldlr is also regulated by other mechanisms.

In the classical bile acid synthesis pathway, CYP7A1, as the rate-limiting enzyme, mediates the transfer of cholesterol to HDL particles for their return to the liver and conversion to bile acids. Cholesterol efflux into bile is enhanced when hepatocytes’ elevated cholesterol levels trigger the production of LXRα, which in turn triggers the activation of the ABCG5/ABCG8 transporter. Inconsistent findings from our investigation included the downregulation of Abcg8 mRNA expression. Another factor that may contribute to cholesterol accumulation in the liver is the reduction of cholesterol efflux into bile caused by downregulating ABCG8 expression. Given its role in cholesterol oxysterol sensing and SREBP-1c activation, hepatic Lxrα mRNA was significantly upregulated by PFUnA at doses of 5 and 10 mg/kg. The transcription factor LXRα favorably regulates Srebp-1c by forming a heterodimer with the retinoid X receptor α. Furthermore, Lxrα mRNA expressions are increased by stimulation of peroxisome proliferator-activated receptor alpha (Pparα), , and Lxrα expression may be increased by endogenous Pparα agonists, such as accumulating fatty acids in the liver. Fatty acid buildup in the liver increases Pparα expression, while Pparα activation activates Lxrα. Furthermore, the synthesis of fatty acids is promoted by the upregulation of Srebp-1c, which is facilitated by the upregulation of Lxrα. Nonetheless, Srebp-1c expression showed no marked difference among the four groups (Figure S2), which is consistent with the liver TG levels found in the study. LXR activation inhibits further absorption of exogenous cholesterol by inducing LDLR ubiquitination and degradation. − In this study, PFUnA downregulated the expression of LDLR, which possibly due to LXRα activation may downregulate LDLR through IDOL-mediated ubiquitination.

In addition to being an indicator of inflammation, Tnf-α is also known to be a risk factor for the onset of NAFLD. Two key pro-inflammatory cytokines, IL-1β and IL-6, exacerbate inflammation and sensitize hepatocytes to TNF-induced liver damage. A member of the ADAMTS family of metalloproteinases, ADAMTS6 plays a role in the remodeling of the extracellular matrix (ECM). ADAMTS6 has been linked in a number of studies to both the development of atherosclerotic plaques and ATH, as well as to the activation of hepatic fibrotic TGF-β. The production of mRNA for the hepatic fibrosis-associated gene Col4a1 causes hepatic fibrosis, which can lead to liver damage. Col4a1, Adamts6, and Il-1β mRNA expression levels were considerably greater in the rat livers of the 10 mg/kg PFUnA group than in the control group, according to our study. Elevated ALT activity and profibrotic gene expression suggest that PFUnA promotes liver inflammation and fibrosis.

In our previous study, PFUnA inhibits the development of pubertal Leydig cells, presumably via promoting autophagy and oxidative stress. Additionally, studies have shown an independent association between the severity of hepatic steatosis and testosterone deprivation. Testicular function is intimately linked to liver development. By increasing MDA levels at doses of 10 mg/kg of PFUnA group and suppressing the synthesis of antioxidants such as SOD1 and CAT, PFUnA caused hepatic oxidative stress. We also observed increased hepatic SOD1 levels at doses of 1 mg/kg PFUnA group; this suggested that rats at 1 mg/kg protected their liver cells from injury by enhancing oxidative stress kinase activity, consistent with the absence of ALT elevation at 1 mg/kg.

Mechanistic studies of PFUnA were conducted by using a variety of experimental approaches. Transcriptomic profiling showed that most downregulated genes were involved in the immune system, cellular enzyme activity, lipid transport, and lipoprotein microparticles, and most upregulated genes were related to lipid compound metabolism, steroid metabolism, the basolateral plasma membrane, cellular protein modifications, and preribosome. This finding implies that PFUnA consumption raises the risk of lipid metabolism disorders and cell homeostasis disruption.

Our in vivo and in vitro studies showed that PFUnA disrupted liver cell development during prepuberty. We measured the mRNA expression of genes related to lipid and cholesterol metabolism in cultured HepG2 cells. The results were almost consistent with those of the in vivo experiments. This also suggests that PFUnA disrupts liver lipid metabolism by regulating cholesterol metabolism and the transport pathway.

Metabolic liver diseases often begin with silent hepatic lipid redistribution and only later progress to steatohepatitis with clinical symptoms. Our doses are supraenvironmental, but we demonstrate that PFUnA has the intrinsic ability to disrupt hepatic metabolic functions. Puberty development is characterized by rapid hormonal changes and metabolic processes. Therefore, low-dose PFUnA exposure, especially during sensitive developmental windows such as puberty, could be a potential environmental risk factor for priming the liver for later metabolic dysfunction. Despite these discoveries, there are a number of issues with our study that need to be addressed in subsequent investigations. The HepG2 cell metabolic profile differs from primary hepatocytes, particularly in basal CYP enzyme activity. Furthermore, our study focused on a single compound (PFUnA). Given that humans are exposed to complex mixtures of PFAS, investigating the potential additive or synergistic effects of PFUnA with other prevalent PFAS (e.g., PFOA, PFOS) would be highly relevant for real-world risk assessment.

In conclusion, our groundbreaking research has demonstrated PFUnA in vivo for the first time in the early stages of NAFLD. We demonstrated that PFUnA causes the phenotype of steatohepatitis by using a rat model treated with PFUnA and comparing the results with those of human hepatic HepG2 cells. By encouraging the release of inflammatory factors, PFUnA not only increased liver weight growth but also sped up the development of steatohepatitis. Crucially, a decrease in the activity of antioxidant enzymes within cells was connected to these effects. In the latter stages of NAFLD, this also shows whether we can create novel medications to enhance mitochondrial activity and boost the antioxidant defense system, fortifying hepatic mitochondria and boosting their resistance to more oxidative damage. The rapid growth of NAFLD worldwide may be prevented by improving the monitoring of PFUnA concentrations in the human or animal body.

Supplementary Material

ao5c07073_si_001.pdf (407.3KB, pdf)

Acknowledgments

Supported by the Natural Science Foundation of Fujian Province (No. 2023J01563) and the Startup Fund for Scientific Research of Fujian Medical University (No. 2021QH1065).

The data supporting this study are available within the manuscript.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07073.

  • Organized specimens and other PCR data (Figures S1 and S2); chemicals, regents, test kits, equipment, software, and service; primer; and antibody information (Tables S1–S4) (PDF)

The authors declare no competing financial interest.

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Associated Data

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Supplementary Materials

ao5c07073_si_001.pdf (407.3KB, pdf)

Data Availability Statement

The data supporting this study are available within the manuscript.

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