2025 Volume 50 Issue 2 Pages 45-55
Perfluorooctane sulfonate (PFOS) is reported to cause hepatotoxicity in animals and humans. However, the underlying mechanism by which it affects organelle toxicity in the liver are not well elucidated yet. This study aimed to investigate the mechanisms underlying PFOS-induced hepatic toxicity, focusing on inflammation, cell death, and autophagy. We established a PFOS-exposed Sprague-Dawley (SD) rat liver injury model by intraperitoneal injection of PFOS (1 mg/kg and 10 mg/kg body weight) every alternate day for 15 days. Our findings indicated that PFOS increased liver weight, caused lipid disorder and hepatic steatosis in rats. Meanwhile, PFOS disrupted the structure of mitochondria, increased accumulation of reactive oxygen species (ROS), repressed superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) levels, and elevated malondialdehyde (MDA) and nitric oxide synthase (NOS) amounts. We found PFOS induced inflammation as evidenced by activation of NOD-like receptor protein 3 (NLRP3), Cleaved cysteine-aspartic acid protease (caspase)1, tumor necrosis factor (TNF)α and interleukin (IL)-1β levels. Moreover, PFOS exposure significantly decreased B-cell lymphoma2 (Bcl2)/Bcl2 associated X (Bax) ratio and increased the protein expression of Cleaved caspase-3. Compared with the control group, PFOS upregulated the protein expression of necroptotic markers and autophagy-related proteins. In conclusion, PFOS induced inflammation, cell death, and autophagy through oxidative stress by ROS overload, thereby providing a mechanistic explanation for PFOS-induced hepatotoxicity.
Perfluorooctane sulfonate (PFOS), an eight-carbon fluorine-saturated compound known for its resistance to degradation and metabolism, is widely used as a raw material for food packaging bags and lubricants (Krafft and Riess, 2015). PFOS can bioaccumulate and biomagnify in both animals and human bodies (Krafft and Riess, 2015). It has been reported that the liver is the organ with the largest accumulation of PFOS, and the most important target organ of PFOS (Cui et al., 2009). Several studies have shown that PFOS accumulation in the liver can result in hepatotoxicity, including hepatic steatosis, hepatocellular hyperplasia, hepatomegaly, and liver peroxisome hyperplasia (Qin et al., 2022; Wan et al., 2012). An epidemiological study has suggested a correlation between the exposure of perfluoroalkyl substances (PFAS) and an increased risk of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) in children (Jin et al., 2020). Although PFOS-induced hepatotoxicity had been acknowledged, the underlying mechanisms were not fully elucidated yet.
Mitochondria are widely acknowledged as a critical cellular organelle, not only for the oxidative phosphorylation but also for metabolism. In the liver, mitochondrial dysfunction disrupts fat homeostasis and contributes to reactive oxygen species (ROS) accumulation, which eventually leads to cytokine overproduction, cell death and lipid peroxidation (Du et al., 2017). It has been observed that animals with NASH are accompanied by mitochondrial integration damage, lower ATP production and more mitochondrial ROS production (Nassir, 2022). ROS can damage to mitochondrial DNA, amplifying oxidative stress and eventually aggravating the oxidative damage to mitochondrial function (Zhang et al., 2019). Although a study indicated that mitochondrial dysfunction is associated with hepatotoxicity of PFOS (Zeng et al., 2021), changes in the structure of mitochondria have not been observed in adult Sprague-Dawley (SD) male rats.
Oxidative stress is recognized to induce by inflammation. In addition, oxidative stress activates the expression of chemokines and proinflammatory cytokines, which led to increased inflammation (Mukhopadhyay et al., 2018). Inflammation that is a hallmark of NASH is important for maintaining critical function of cells (Bessone et al., 2019). NOD-like receptor protein 3 (NLRP3) inflammasome which can trigger inflammation and pyroptosis. Previous research has shown that excessive production of ROS stimulates pyroptosis by activating the NLRP3 inflammasome (Li et al., 2022). NLRP3-mediated pyroptosis has been implicated in various organ injuries, including liver damage (Li et al., 2022). Recent research found that in high-fat diet (HFD)-fed mice, PFOS treatment (5 mg/kg/day) can dramatically aggravate inflammation in the liver via stimulating NLRP3 and cysteine-aspartic acid protease (caspase)1 expression (Qin et al., 2022). Though PFOS-induced inflammation has been acknowledged as a key factor for its hepatotoxicity, whether ROS is involved in inflammation after PFOS treatment in rats was not fully elucidated yet.
Apoptosis and necrosis are two key forms of cell death that are pivotal in directing the severity and outcome of liver injury. Additionally, apoptosis and necroptosis, triggered by chronic liver diseases such as alcoholic steatohepatitis, can initiate hepatic regeneration, inflammation, and fibrogenesis (Schwabe and Luedde, 2018). Excessive ROS levels inflammation may can trigger cell damage and/or death, leading to hepatocellular damage and elevated liver enzymes (Bessone et al., 2019; Wu et al., 2022). Our previous study demonstrated that PFOS increases ROS levels and induces apoptosis in the kidney tissue of rats (Tang et al., 2022). Additionally, the production of ROS can cause the necroptosis of hepatocytes in vivo study (Liao et al., 2020). Sodium p-perfluorous nonenoxybenzene sulfonate (OBS) used as a substitute for PFOS induced receptor-interacting protein (RIP)1/RIP3-dependent programmed necroptosis in HepG2 cells (Ye et al., 2024). However, whether PFOS induces necroptosis in the liver of rats is unknown. It is reported that multiple types of cell death modes can likely coexist, and the death of different liver cell populations may contribute to liver injury. Therefore, clarifying whether PFOS induces both apoptosis and necroptosis simultaneously in a rat liver injury model is of considerable scientific significance.
Autophagy is a cellular process that regulates the degradation and recycling of damaged or unnecessary organelles. The interaction between hepatic autophagy and programmed cell death (including pyroptosis, apoptosis, and necroptosis) plays a significant role in both physiological and pathological processes (Shojaie et al., 2020). In addition, excessive ROS production in mitochondria and hepatic cell death can trigger autophagy (Shan et al., 2018; Ueno and Komatsu, 2017). A previous study showed that the ROS-triggered autophagy increased after PFOS exposure in human embryo liver L-02 cells (Zeng et al., 2021). However, it remains unclear whether ROS-triggered autophagy is involved in PFOS-induced hepatotoxicity in vivo.
In this study, we demonstrated that PFOS promoted inflammation, cell death, and autophagy through oxidative stress by ROS overload in a rat model, which might provide new insights into the study of the hepatotoxicity of PFOS.
Male SD rats (160 ± 5 g) were procured from Zhejiang Laboratory Animal Center (Hangzhou, China). They were freely allowed food and water, and housed at room temperature. Animals received humane care in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experimental procedures were executed based on Experimental Animal Ethics Committee of Hangzhou Medical College’s guidelines (approval number: 20200178).
Rat allocation was performed as follows (n = 6): control group (0 mg/kg PFOS), 1 mg/kg PFOS group and 10 mg/kg PFOS group. The dose, route of administration, and dose regimen of PFOS (Energy Chemical, Beijing, China) were selected based on previous studies (Austin et al., 2003; Tang et al., 2022). PFOS (CAS 2795-39-3, 98% purity, FW538.22, Energy Chemical, China) was dissolved in 0.9% normal saline to prepare solutions of 2 and 0.2 mg/mL PFOS, respectively. Rats in the PFOS groups received intraperitoneal injections of PFOS every other day for 15 days. At the same time, control group received normal saline. Fifteen days later, rats were anesthetized, followed by sacrificing to minimize potential pain and distress. Blood and the liver samples were harvested for the subsequent experiments.
Determination of body and liver weightsBody weight was monitored every other day, and livers were immediately weighed on day 15. Based on the formula of liver weight/body weight × 100%, liver index (%) was calculated.
Serum biochemical assaysBlood samples (1 mL) were stood, followed by centrifugation at 1000 g for 10 min. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents in serum were measured using an automatic biochemistry analyzer (Beckman).
The activities of superoxide dismutase (SOD), nitric oxide synthase (NOS), and glutathione peroxidase (GSH-PX), the concent of and malondialdehyde (MDA) were measured by the assay kits (Nanjing Jiancheng, China).
Histological analysis and steatosis scoreLiver samples were first fixed with 4% paraformaldehyde for 8 hr, followed by embedding in paraffin at 60°C. Samples were sectioned (5-μm-thickness) and stained with hematoxylin and eosin (H&E) for assessing histopathology. Images were photographed under an inverted phase-contrast microscope (Leica Microsystems, Germany).
Steatosis scoring was performed as previously depicted (Han et al., 2018). The steatosis score was categorized into 5 grades according to the severity and extension of hepatic steatosis. Briefly, 0: absent of vacuolation; 1: 2 or 3 vacuoles per hepatic cord per lobule; 2: less than 50% of the lobule exhibits fatty vacuolation; 3: more than 50% of the lobule exhibits fatty vacuolation; 4: nearly the entire lobule shows fatty infiltration.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining assayTUNEL staining was executed to detect apoptotic cells in liver tissue of SD rats. Liver paraffin sections were stained in accordance with the instructions. The sections (5-μm-thickness) were mixed with TUNEL reaction mixture for 1 hr, washed with phosphate-buffered saline (PBS), and incubated with 4',6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime, 1:1000 dilution) solution for 10 min in the dark. Lastly, images were photographed under an inverted fluorescence microscope. The TUNEL-positive hepatocytes were analyzed and quantified by five non-repeating random areas.
ROS expression measurementROS production in liver tissues was analyzed with a DHE probe using a frozen sections ROS testing kit (Genmed Scientifics, Shanghai, China). At room temperature, the frozen sections were first spin-dried for 5 min and then washed with Reagent A, followed by dyeing with staining solution at 37°C for 30 min. Then, the slices were washed three times with PBS and dyed with DAPI (C1002, Beyotime, 1:1000 dilution) for 10 min. After that, sections were covered with Mounting Medium, antifading and then photographed under a fluorescence microscope.
Transmission electron microscopeLiver tissues of three groups were harvested, quickly fixed in the electron microscope fixing solution, and washed. Specimens were fixed and then dehydrated. After that, samples were first embedded in poly/bed-812 epoxy resin at 37°C overnight, then sectioned at 70 nm thickness. The 70 nm sections were prepared and stained using uranium-lead double staining. Images were photographed under a transmission electron microscope (Hitachi, Japan).
Immunohistochemical analysisThe sections were blocked with 3% BSA and incubated with primary antibody, CD68 antibody (ab125212, Abcam, 1:600 dilution) overnight at 4°C. Then, the sections were washed, mixed with secondary antibody (sc-3753, Santa, 1:500 dilution) for 2 hr and visualized with substrate diaminobenzidine (DAB) in the dark. Images were photographed via a microscope (Leica, Germany). The positive cells were analyzed and quantified by Image-Pro Plus software (Media Cybernetics, United States).
Enzyme-linked immunosorbent assay (ELISA)Liver tissues were minced in pieces, followed by homogenizing on ice, then homogenates were centrifuged at 1000 g for 10 min. To detect protein levels of IL-1β and TNFα in liver tissues, the clear supernatant extract was used with ELISA in accordance with the instructions (Anogen, Mississauga, Ontario, Canada).
Western blottingProteins were extracted and collected for western blotting. Primary antibodies: NLRP3 (19771-1-AP, Proteintech), Cleaved caspase1 (22915-1-AP, Proteintech), p-RIP1 (PA5-105363, Invitrogen), p-RIP3 (PA5-105701, Invitrogen), p-mixed the lineage kinase domain-like (MLKL) (PA5-105678, Invitrogen), B-cell lymphoma2 (Bcl2) (26593-1-AP, Proteintech), Bcl2 associated X (Bax) (ABP50752, Abbkine), Cleaved caspase-3 (9664S, Cell Signaling), putative kinase (PINK)-1 (ab186303, Abcam), parkin (ab77924, Abcam), light chain (LC)3II/I (14600-1-AP, Proteintech) and GAPDH (60004-1-1G, Proteintech) were used. All primary antibodies were diluted to 1:1000 except for GAPDH (1:10000 dilution). Two types of secondary antibodies, goat anti-mouse antibody (A21010, Abbkine, 1:10000 dilution) and goat anti-rabbit antibody (A21020, Abbkine, 1:10000 dilution), were executed to incubate with the membranes for 1 hr at room temperature. We used Quantity One software (version 4.2.2, Bio-Rad Laboratories, USA) to analyze the density of the products.
Statistics analysisData analyses were conducted using GraphPad Software (GraphPad Software, Inc, USA) and presented as mean ± standard deviation with six rats in each group to ensure their reliability. ANOVA followed by Tukey’s post-hoc test was executed for analyses. P value less than 0.05 means the data were significant.
Body weights recorded every other day are presented in Fig. 1A. The body weight in the PFOS treatment groups was relatively lower than the control group on day 15. Meanwhile, 10 mg/kg of PFOS dramatically increased the liver index in contrast with that of the controll group (P < 0.05; Fig. 1B). To ascertain whether PFOS treatment could cause lipid disorder, the levels of TC, TG, HDL and LDL in serum were analyzed. In contrast with the 0 mg/kg PFOS group, TC, TG, HDL and LDL content levels were all repressed in the 10 mg/kg PFOS treatment group (P < 0.05; Fig. 1C-F). Additionally, for the 1 mg/kg PFOS group, as shown in Fig. 1D and E, it also downregulated the TG and HDL content. Serum ALT and AST levels in the 1 mg/kg PFOS group showed no significant difference with the 0 mg/kg PFOS group (Fig. 1G and H). However, the 10 mg/kg PFOS treatment markedly elevated the serum ALT and AST (P < 0.05; Fig. 1G and H). Moreover, H&E staining results showed that PFOS caused hepatocellular irregularity and steatosis (Fig. 1I), and increased the steatosis score compared to the group without PFOS treatment (Fig. 1J). These results imply that PFOS increased liver weight, and caused lipid disorder and hepatic steatosis in SD rats.
PFOS increased liver weight, caused lipid disorder and hepatic steatosis in SD rats. (A) Body weights of rats in three groups were recorded every other day for 15 days. (B) liver index, the percentage of liver weight to body weight, was assessed on day 15. (C-F) The TC, TG, HDL and LDL contents in serum were assessed on day 15. (G and H) The ALT and AST in serum of rats in three groups. (I) Hepatic steatosis was revealed by immunohistochemical analysis. White arrows show hepatic cell edema and degeneration. Black arrows show fatty vacuolation. (J) The steatosis score after exposed to different doses of PFOS. Values are mean ± SD, n = 6. *P < 0.05, and **P < 0.01 vs 0 mg/kg group. Bar = 100 µm.
To probe the effect of PFOS on mitochondrial structure of the liver in SD rats, a transmission electron microscope was used. As shown in Fig. 2A, in the PFOS treatment groups, mitochondria appeared to have lost cristae and to be swollen with vacuolar structure, rough endoplasmic reticulum (ER) was decreased and distended, and normal mitochondria-associated endoplasmic reticulum membrane (MAM) structures were disrupted. Mitochondrial dysfunction generates excesses of mitochondrial ROS that cause cellular damage. To further scrutinize the association of PFOS with mitochondrial dysfunction, we first probed the levels of ROS in liver tissues of rats. As shown in Fig. 2B and C, PFOS treatment dramatically elevated ROS levels (P < 0.01). MDA and NOS are two critical markers of oxidative stress, and SOD and GSH-PX are two markers of antioxidant enzymes. It was shown that 10 mg/kg PFOS remarkably decreased the activities of SOD and GSH-PX in contrast with the group without PFOS treatment (P < 0.05, Fig. 2D and E), while there were no notable differences across the PFOS groups. Furthermore, MDA level and activity of NOS were significantly increased in the higher-dose PFOS group (P < 0.05, Fig. 2F and G). These data showed that PFOS caused mitochondrial dysfunction and induced oxidative stress.
PFOS impaired mitochondrial structure and caused oxidative stress injury. (A) The inner structures of mitochondria, ER, and MAM in hepatic cells were impaired after PFOS treatment (M: mitochondria; ER: endoplasmic reticulum; MAM: mitochondria-associated endoplasmic reticulum membrane), Bar = 0.2 μm, 0.5 μm, 2 μm. (B and C) ROS production in liver tissues was analyzed via an immunofluorescent staining assay and quantitative analysis, Bar = 100 μm. (D and E) The activities of SOD and GSH-PX in three groups. (F and G) The level of MDA and the activity of NOS in three groups. Values are mean ± SD, n = 3 or 6. *P < **P < 0.01 and ***P < 0.001 vs 0 mg/kg group.
We investigated PFOS’s influences on hepatic inflammation in SD rats. As presented in Fig. 3A and B, expression of macrophage marker CD68 was significantly increased in the livers of the 1 mg/kg and 10 mg/kg PFOS groups compared with the 0 mg/kg PFOS group (P < 0.05). Consistently, compared with 0 mg/kg PFOS, higher expression of IL-1β and TNFα was also observed in the PFOS treatment groups (P < 0.05; Fig. 3C and D). To examine whether the NLRP3 signaling pathway is involved in PFOS-induced hepatotoxicity, western blotting assays were used. As demonstrated in Fig. 3E and F, we found NLRP3 and Cleaved caspase1 expression in the PFOS groups was distinctly enhanced than that of the control group (P < 0.05). These data demonstrated that PFOS activated hepatic inflammation through the NLRP3 signaling pathway, which plays an important role in hepatotoxicity.
Hepatic inflammation activation took part in hepatotoxicity induced by PFOS. (A and B) Immunohistochemistry staining in liver showed the effect of PFOS on Kupffer cell accumulation. Red arrows show CD68-positive cells. (C and D) The levels of pro-inflammatory cytokines IL-1β and TNFα in liver were assessed by ELISA. (E) Protein levels of NLRP3 and Cleaved caspase1 in liver were assessed by western blotting. (F) Quantitative analysis of NLRP3 and Cleaved caspase1 proteins. Values are mean ± SD, n = 3. *P < 0.05, and **P < 0.01 vs 0 mg/kg group. Bar = 100 µm.
It is well known that excessive ROS production in mitochondria can trigger apoptosis and necroptosis. TUNEL assay was used to ascertain the influences of PFOS on cell death. As shown in Fig. 4A, there was a higher fraction of dead cells which were marked on day 15 following PFOS treatment than in the 0 mg/kg PFOS group in liver tissues. The quantification revealed that PFOS groups greatly increased the number of TUNEL-positive cells relative to the group without PFOS treatment (P < 0.01, Fig. 4B). Meanwhile, we demonstrated that Bcl-2/Bax ratio was significantly lower and Cleaved caspase-3 levels were upregulated by 10 mg/kg PFOS treatment (P < 0.05; Fig. 4C-E). We then measured the levels of necroptotic markers (RIP1, RIP3 and MLKL) in liver tissues. It was indicated that PFOS not only elicited the phosphorylation (p) of these proteins, but also increased the expression of p-RIP1, p-RIP3 and p-MLKL dramatically (P < 0.05; Fig. 4F-I). The outcomes suggest that PFOS may lead to apoptosis and necroptosis via the accumulation of ROS, thereby aggravating the development of hepatotoxicity.
PFOS induced cell death in the liver of rat. (A and B) TUNEL staining of liver tissues, and the quantification of TUNEL-positive cells. (C-E) Protein levels of apoptosis markers (Bax, Bcl-2 and Cleaved caspase-3) in liver tissues were measured by western blotting and quantitative results. (F-I) Protein levels of necroptotic markers (p-RIP1, p-RIP3, and p-MLKL) in liver tissues were measured by western blotting and quantitative results. Values are mean ± SD, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 vs 0 mg/kg group. Bar = 100 µm.
The occurrence of both apoptosis and necroptosis is usually accompanied by autophagy. We further observed the expression of autophagy-related proteins (PINK-1, parkin and LC3II/I) could be stimulated by PFOS treatment (P < 0.01; Fig. 5A-D).
PFOS induced autophagy. (A) Protein levels of autophagy-related proteins (PINK-1, parkin and LC3II/I) in liver tissues were determined by western blotting. (B-D) Quantitative analysis of PINK-1, parkin and LC3II/I proteins. Values are mean ± SD, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 vs 0 mg/kg group.
PFOS, one member of the PFAS family, has been shown to be hepatotoxic, nephrotoxic and cardiotoxic (Qin et al., 2022; Tang et al., 2017., Tang et al., 2022). Moreover, epidemiological studies showed PFOS elevated liver function damage both in children and adults (Jin et al., 2020; Bassler et al., 2019). A previous study found that the molecular mechanisms involved in PFOS causing hepatotoxicity are mainly referring to fat metabolism, oxidative stress, and cell cycle (Zeng et al., 2019). Since excessive ROS and inflammation can stimulate cell death, which in turn can trigger autophagy, we further supposed that inflammation, cell death, and autophagy through oxidative stress by ROS overload could be major mechanisms of PFOS-induced hepatotoxicity.
PFOS was reported to decrease body weight but boost liver weight in an animal model (Wan et al., 2012). Consistent with previous research, this study found that PFOS treatment decreased the body weight to some extent, and significantly increased the liver index in a dose-dependent manner. The European Food Safety Authority found that PFOS elevated serum TC levels in humans (Behr et al., 2020). However, in an animal study, serum cholesterol levels were confirmed to be decreased after PFOS treatment (Elcombe et al., 2012). While another study reported that oral gavage at 10 mg/kg PFOS increased liver TG load in mice (Das et al., 2017). Unfortunately, the effects of PFOS on TG and TC concentrations in SD rats are inconclusive yet. In the current work, the levels of TG, TC, LDL and HDL in serum were decreased in the higher-dose PFOS group, which was in line with previous research (Wang et al., 2014). These effects in SD rats are inconsistent with those in humans and mice, which might be due to species-specific differences. The results in this study revealed that PFOS increased liver weight and caused lipid disorder in SD rats.
An early study reported a borderline positive correlation between serum PFOS concentration with serum levels of ALT in 2,200 individuals (Lin et al., 2010). However, another research reported that there was no association between PFOS and liver enzymes in a sample of Chinese workers and residents (Wang et al., 2012). Current studies on the relationship between serum PFOS concentration and serum liver enzymes are inconsistent. We found that serum ALT and AST levels were markedly elevated in the higher-dose PFOS group, which is in agreement with a previous report that SD rats daily given 10 mg/kg PFOS by gavage exhibited impaired liver function (Han et al., 2018). Secondly, pathological changes were observed to identify hepatotoxicity in rats caused by PFOS. In this study, we found that 1 and 10 mg/kg PFOS treatment for 15 days greatly increased hepatic steatosis in rats, which was consistent with a previous study in mice orally exposed to 1, 5 and 10 mg/kg dose of PFOS for 14 days (Jiang et al., 2022).
The liver can provide bodily energy through the abundant mitochondria in its cells. Maintenance of normal mitochondrial morphology and mitochondria-associated ER membrane ensures adequate ATP production and cellular function (Daum et al., 2013). Though a study showed that mitochondrial dysfunction was associated with the liver toxicity of PFOS (Han et al., 2018), the structure of liver mitochondrial had not been observed in adult SD male rats. In this study, electron microscopic observation of the liver tissue found that PFOS led to damage of mitochondria, ER and MAM structures. As is well known, mitochondria are the primary source of ROS, and mitochondrial dysfunction disrupts fat homeostasis and contributes to ROS accumulation. In turn, excessive ROS can damage the structure of mitochondrial membranes and impair mitochondrial function (Zhang et al., 2019). ROS are highly reactive and primarily target polyunsaturated fatty acids (PUFAs). Arachidonic acid (a type of PUFA) undergoes peroxidation, ultimately forming malondialdehyde (MDA). MDA is widely recognized as a biomarker of oxidative stress, particularly of lipid peroxidation, and is likely correlated with the accumulation of lipid droplets (Tsikas, 2017). Intracellular antioxidant enzymes are essential for maintaining the balance of intracellular ROS (Ye et al., 2024). SOD and GSH-Px are two key antioxidant enzymes that eliminate free radicals and lipid peroxides (Wu et al., 2022). Consistent with an earlier report, our results showed that ROS production in liver tissues was indeed elevated when exposed to PFOS (Han et al., 2018). Additionally, we further demonstrated that relatively high concentration of PFOS overtly prohibited SOD and GSH-PX activities, and enhanced the levels of MDA and NOS. These results revealed that a relatively higher dose of PFOS (10 mg/kg) could induce mitochondrial dysfunction, thereby inducing oxidative stress.
Inflammation in liver is important for the development of hepatocyte death (Bassler et al., 2019). In addition, inflammation contributes to non-alcoholic steatohepatitis characterized by liver steatosis (Teixeira et al., 2023). Oxidative stress has been considered as a potential cause in steatosis-related inflammatory response. Oxidative stress and consequent lipid peroxidation can stimulate inflammatory process, resulting in an elevation in inflammatory cytokines such as TNFα, IL-6, and IL-1 (Hussain et al., 2016). A study reported that PFOS exposure remarkably increased the amounts of TNFα and IL-6 in animals (Han et al., 2018). NLRP3 takes part in inflammation activation and pyroptosis, which is activated by production of ROS. NLRP3 was reported to participate in the proteolytic maturation of caspase1, which eventually cleaves and activates pro-IL-1 to mature and active IL-1β (Fu and Wu, 2023). The release of proinflammatory cytokines like IL-1 and TNFα leads to proinflammatory responses and the recruitment of neutrophils and monocytes, amplifying liver tissue injury. Growing evidence has linked NLRP3-mediated inflammation and pyroptosis to liver damage and fibrosis in conditions such as nonalcohoic steatohepatitis and drug-induced liver injury (Wree et al., 2018). Recent research found that mice treated with 2.5 mg/kg body weight (BW) of PFOS via daily gavage for 6 weeks exhibited hepatocyte pyroptosis as demonstrated by activation of the NLRP3 (Ren et al., 2024). Our results demonstrated that NLRP3 participated in PFOS-activated hepatic inflammation in SD rats.
Liver-resident and recruited immune cells release pro-inflammatory signals that promote hepatocyte death, which in turn triggers a damaging feedback loop of inflammation and cell death (Brenner et al., 2013). It has been reported found that the accumulation of many toxicants in the liver can cause apoptosis and necroptosis of hepatocytes (Wang et al., 2022; Zhang et al., 2020). Apoptosis is a type of programmed cell death, which is critical for maintaining normal cell turnover. Cellular apoptosis could be caused by oxidative stress via the mitochondria-dependent pathways (Sinha et al., 2013). The Bcl2 and Caspase 3 are the key mediators of apoptosis (Tang et al., 2022). PFOS at a single dose of 1 or 10 mg/kg body weight for 28 consecutive days has been reported to trigger apoptosis in rats (Han et al., 2018). This study found that compared with the control group, PFOS groups significantly decreased the ratio of Bcl2 to Bax and dramatically elevated cleaved-caspase-3 production. These results suggest PFOS ultimately led to apoptosis in rats. Necroptosis is another kind of programmed cell death that is generally modulated through activating RIP1, RIP3 and MLKL (Khodayar et al., 2021). A previous study has indicated that in hepatotoxic mice, necroptosis markers (RIP1, RIP3, and MLKL) were activated and their expression was significantly elevated (Khodayar et al., 2021). Another study found that hepatic RIPK3 correlates with NAFLD severity, playing a key role in managing liver lipid metabolism (Afonso et al., 2021). A recent study reported that treatment with 0.05 and 20 uM PFOS for 48 hr inhibited necroptosis in hepatocellular carcinoma cells (Hong et al., 2024). Interestingly, our study found that PFOS treatment could increase the amounts of p-RIP1, p-RIP3, and p-MLKL distinctly, suggesting that PFOS elicited necroptosis in hepatotoxic rats. These findings indicate that apoptosis and necroptosis may play critical roles in PFOS-induced hepatotoxicity.
Additionally, autophagy, as a crucial biological process at the cellular level has been reported to be related to liver diseases, such as hepatitis and liver cancer, and of course hepatotoxicity (Shan et al., 2018). PINK1-parkin-involved mitophagy is the most typical signaling pathway (Shan et al., 2018). In impaired mitochondria, both PINK-1 and parkin were significantly enriched. Similarly, this study showed that PFOS dramatically promoted the expression of PINK-1 and parkin in hepatotoxic rats. Not only that, LC3II/I, as another important autophagy marker, was also elevated by PFOS treatment. Therefore, we concluded that PFOS was also an inducer for autophagy in hepatotoxic rats.
ConclusionsIn summary, we uncovered that inflammation, cell death, and autophagy through oxidative stress by ROS overload might be important aspects of hepatotoxicity caused by PFOS. These might be helpful in revealing the sensitive targets posed by PFOS pollution, and provide new ideas for the prevention and treatment of organ toxicity caused by PFOS.
We acknowledged the strong support of Affiliated Xiaoshan Hospital, Hangzhou Normal University, The First People's Hospital of Yuhang District and Hangzhou Medical College.
FundingThis work was supported by Science and Technology Plan Project of Hangzhou (Grant Number 20201231Y118), Medical Health Science and Technology Program of Zhejiang (Grants Numbers 2024KY1443, 2024KY1448), Medical Health Science and Technology Program of Hangzhou (Grants Numbers B20210454), the biomedical and Health Industry Development Support Science and Technology Project of Hangzhou (Grants Numbers 2022WJC139, 2022WJC234)
Conflict of interestThe authors declare that there is no conflict of interest.