• PFAS exposure activates the H₂O₂ screening system, mainly in the caudal kidney.

Among the anthropogenic contaminants, per- and polyfluoroalkyl substances (PFAS) have garnered the attention of the scientific community and public opinion in the last decade due to their widespread use and high persistence and toxicity in the environment (Cousins et al., 2020), which can also increase in relation to Climate change (Bortot et al., 2025; Cunha et al., 2025). As they are used in a wide range of fields, from carpeting to dental floss (Glüge et al., 2020), PFAS have a global distribution and are even present in Antarctica (Xie et al., 2022). The amphiphilic structure of PFAS, consisting of a polar functional group and a hydrophobic fluorinated tail, endows these substances with a high affinity for proteins, such as liver fatty acid-binding proteins and serum albumin. As a result, PFAS can bioaccumulate within organisms and be transferred along the trophic chain (Ferrario et al., 2022; Lewis et al., 2022; Pietropoli et al., 2025). Regarding human health, some of the main studied effects of exposure to PFAS include alteration of immune function and interference with the efficacy of vaccines, liver and kidney diseases, including cancer, diabetes due to alteration of lipids and insulin concentration, infertility and growth failure (Bonato et al., 2020; Fenton et al., 2021). The manufacture and use of the most well-known PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), have been restricted in many countries precisely because of their significant potential risks to human health and the environment (Louisse et al., 2020).

In 2013, a significant industrial PFAS contamination of surface water and groundwater was discovered in the Veneto region, Italy (Giglioli et al., 2023). According to annual monitoring data from the Regional Agency for Environmental Prevention and Protection of Veneto (ARPAV) (ARPA Veneto, 2025), the presence of PFAS in freshwater streams continues to be assiduous, making it necessary to investigate the ability of organisms to cope with the stress induced by prolonged exposure to PFAS (Vaccari et al., 2024).

Among the species currently subjected to this anthropogenic impact is Squalius cephalus, also known as European chub, a pelagic freshwater fish abundant throughout Europe, commonly used as a target species in ecotoxicological studies (Molbert et al., 2019; Sunjog et al., 2019; Staszny et al., 2021). S. cephalus specimens typically live 15 years, with males becoming fertile at 2–4 years and females at 4–6 years (Nyeste et al., 2024).

Our previous study showed that PFAS contamination in rivers of the Veneto region led to a significant increase in transcript levels of gpx-4 and sod-2, two genes encoding mitochondrial antioxidant enzymes, in the liver of S. cephalus and another species, Padogobius bonelli. The results suggested that oxidative stress, which could lead to lipid peroxidation due to chronic PFAS exposure, was controlled in this organ by the antioxidant system at the mitochondrial level (Piva et al., 2022). The adequate protection of lipids from PFAS was also confirmed by analyses performed on the kidney of S. cephalus, in which, however, protein oxidation was detected also at low PFAS concentrations (Pacchini et al., 2025b). In this case, the gene encoding the isoform 4 of peroxiredoxins (prdx-4) appeared not to be involved in the antioxidant defences, probably carried out by other components of the antioxidant system. Further analyses also revealed high stress levels in the muscle and blood of S. cephalus and P. bonelli (Schumann et al., 2024), underscoring the need to extend the investigation of responses against PFAS to other organs and genes.

Glutathione peroxidases (GPxs) and catalase (CAT) act mainly in H₂O₂ scavenging. GPxs reduce organic and inorganic peroxides to hydroxyl compounds, using glutathione (GSH) or other equivalents as reducing factors. Among the known eight GPx isoforms in humans, three are monomeric, i.e. GPx-4, -7 and -8, and the remaining five are homotetrameric. GPx-3, -6 and -7 are the only extracellular isoforms; the others are intracellular. Among the latter, isoform 4 is the only one present in mitochondria. The first four isoforms and GPx-6 are selenium (Se)-dependent, as they present a selenocysteine residue encoded by the stop codon TGA (Brigelius-Flohé and Maiorino, 2013; Ferro et al., 2020; Pacchini et al., 2025a). While GPx-4 can reduce complex fatty acid, phospholipid, and cholesterol hydroperoxides, including those inside membranes, GPx-1, -2, and -6 are supposed to react with less complex, soluble, but also small fatty acid hydroperoxides (Trenz et al., 2021). Unlike GPxs, CAT exists as a single isoform in animals that can be distinguished into three classes based on its sequence and structural differences. The monofunctional heme-containing enzyme is the most widespread CAT in all aerobic organisms. The bifunctional CAT-peroxidase belongs to the second class, which also contains the heme group but is relatively less abundant in nature and closely related to plant peroxidases. The third class belongs to the Mn-containing CAT group that lacks the heme group (López et al., 2024). CAT is a tetrameric protein that can cleave two H₂O₂ molecules into two water molecules and one oxygen molecule, acting mainly within peroxisomes. CAT has been reported to be implicated in mutagenesis, inflammatory conditions, and the suppression of apoptosis, all of which are known to be associated with oxidative stress conditions (Nandi et al., 2019).

The present work aims to gain a deeper understanding of how the components of the antioxidant system interact to counteract the adverse effects of PFAS in S. cephalus on the liver and caudal kidney. S. cephalus specimens were collected from four rivers characterised by different concentrations of total dissolved PFAS, from <10 ng/L (control site) to over 1,000 ng/L (highly-polluted site). The antioxidant molecular markers considered in this study were GPx isoforms 1 and 4 and CAT. Both gene expression and enzyme activity were assessed in the liver and caudal kidney, organs known for their roles in the bioaccumulation of pollutants in fish and detoxification processes, as they filter contaminants from the bloodstream and metabolise them for elimination, primarily via urine (Collard et al., 2018; Macorps et al., 2022).

Additionally, to better study the acclimatisation to the environment, the morphological characteristics of the fish were also examined, as well as the development of their organs, calculating the hepatosomatic index (HSI), Fulton’s condition factor (FCF) and the spleen somatic index (SSI). Finally, liver and caudal kidney tissues were chemically analysed to verify if PFAS accumulation increased proportionally to the increase in PFAS concentration in the environment, and were examined by hematoxylin and eosin staining to highlight cellular damage and lipid content.

Most research on PFAS focuses on human health (Biggeri et al., 2024), while little is known about their effects on freshwater fauna. Given that water is the primary polluted matrix in Veneto (Biggeri, 2024; Vaccari et al., 2024), investigating the role of chronic exposure to PFAS on the physiology of S. cephalus, an endemic and cosmopolitan fish species in the region, may be fundamental to better understanding their impact on river ecosystems.

Previous studies have shown that S. cephalus can activate a range of physiological responses at both cellular and systemic levels in response to PFAS exposure (Piva et al., 2022; Schumann et al., 2024; Pacchini et al., 2025b). In the present study, in addition to evaluating three somatic indices to obtain a general overview of the wellbeing of the sampled S. cephalus specimens, we investigated the antioxidant responses of this freshwater fish that counteract oxidative stress induced by H₂O₂ overproduction. To this aim, we examined the induction of CAT and Se-GPx, both at transcriptional and enzymatic activity levels, in the liver and caudal kidney, two organs involved in the accumulation and detoxification of xenobiotics.

Regarding the HSI, we observed a clear increase in liver size in fish from medium- and highly-polluted sites. This finding is consistent with previous evidence linking liver enlargement in S. cephalus to total PFAS concentrations (Piva et al., 2022) and, in our case, specifically to PFOS and PFDA, the predominant compounds detected in our analyses. Hepatic PFOS levels reached 55.40 μg/L and 72.78 μg/L at the medium- and highly-polluted sites, respectively, while PFDA concentrations were 3.31 μg/L and 21.67 μg/L. These concentrations are in line with the liver being a primary PFOS target organ (Wang et al., 2022) and fall within ranges associated with hepatic steatosis in fish and rodents chronically exposed to PFAS (Attema et al., 2022; Cheng et al., 2016). Such steatosis, driven by stress-induced lipid accumulation in hepatocytes, may reduce PFAS interactions with cellular proteins by modifying the hydrophilicity of the intracellular environment and promoting non-specific binding to lipids (Yoo et al., 2009; Qin et al., 2023). Moreover, PFAS can disrupt lipid metabolism through activation of PPARα and PPARγ, further enhancing lipid deposition (Li et al., 2024; Wang Q. et al., 2023; Johnson et al., 2024), while lipid droplets may confer a protective effect by sequestering reactive lipid peroxidation products and limiting oxidative damage (Martinez et al., 2023). The liver is not the only organ affected by weight increases following PFAS exposure, as indicated by the FCF index, which in our study also revealed higher total body weight in S. cephalus from medium- and highly-polluted sites.

Our histological analyses revealed lipid accumulation not only in the liver but also in the caudal kidney. While there is limited evidence in fish regarding renal lipid deposition linked to PFAS, human studies have associated chronic kidney disease with PFAS exposure (Conway et al., 2018), and this condition is known to cause lipid dysmetabolism and lipid droplet accumulation in the kidney (Mitrofanova et al., 2023). Supporting this hypothesis, we observed downregulation of prdx-4 mRNA expression in the caudal kidney of S. cephalus chronically exposed to PFAS (Pacchini et al., 2025b). Since this antioxidant enzyme inhibits lipid accumulation in cells (Yamada and Guo, 2018), its reduced expression may reflect a cellular response facilitating lipid deposition in the kidney. Similarly, Kim et al. (2023) demonstrated that reduced antioxidant enzyme activity promotes lipid droplet formation under chemical stress.

The SSI index results showed the highest values in fish from the low-polluted site and the lowest in those from the highly-polluted site. The spleen is particularly interesting because it plays a crucial role in regulating the immune system. Previous studies on mice exposed to PFOS have evidenced a decrease in spleen weight, which was associated with an increase in liver weight (Wang et al., 2011), suggesting an adverse effect of this PFAS on the immune system. Spleen immunosuppression was also highlighted in zebrafish exposed to PFOA (Zhong et al., 2020). The spleen plays a crucial role in erythropoiesis, the process of producing red blood cells. However, in fish from medium- and highly-polluted sites, this function may be predominantly carried out by the head kidney (Chen et al., 2013; Witeska, 2013). The increase in spleen weight observed at the low-polluted site is likely due to enhanced erythrocyte production or activation of the immune response. This response may be necessary when evaluating prolonged exposure to PFAS, even at low concentrations. Based on these considerations, it is plausible that the spleen enlarges at the low-polluted site as a primary defence mechanism against the effects of PFAS. When these effects become too excessive for the organism (medium- and highly-polluted sites), the spleen goes under stress, decreasing its mass. Therefore, the spleen appears to play a secondary role in defending against PFAS when their concentrations are high, leaving the liver and the body’s general lipid accumulation to deal with the harmful effects of PFAS.

We observed a limited activation of the studied antioxidant enzymes in the liver. No change in CAT activity is consistent with previous findings in rainbow trout liver (Tilton et al., 2008) and in primary cultured hepatocytes of freshwater tilapia (Liu et al., 2007) exposed to PFOA, as well as in salmon hepatocytes exposed to perfluorooctane sulfonamide (PFOSA) (Olufsen and Arukwe, 2015). Similarly, a non-activation of GPx activity was known in Atlantic salmon hepatocytes exposed to PFOSA (Wågbø et al., 2012) and in hepatocytes of freshwater tilapia exposed to PFOA (Liu et al., 2007). This result can be explained by the fact that other components of the antioxidant system may have been activated. It is known, for example, that in the cell, in addition to CAT and GPx, the Prdxs, as mentioned above, also operate as H₂O₂ scavengers (Al-Asadi et al., 2019).

In contrast, the caudal kidney showed induction of both CAT and Se-GPx. This finding is expected, given that the kidney is the second most important organ for PFAS accumulation (Savoca and Pace, 2021), as also confirmed by our chemical analyses, which demonstrated increasing levels of PFOS and PFDA in fish moving from the control site to the highly-polluted one. Specifically, PFOS levels reached 70.54 μg/L and PFDA 32.90 μg/L in the caudal kidney of fish from the highly-polluted site. Similar trends of PFAS bioaccumulation, with PFOS predominance in various organs, have been reported in freshwater fish (Pivonkova et al., 2020; Smith et al., 2021). PFDA has been shown to induce oxidative stress and mitochondrial dysfunction in cellular and zebrafish embryo models, impairing fatty acid β-oxidation and increasing reactive oxygen species production (Widhalm et al., 2024; Piva et al., 2022). These effects support the mechanistic plausibility of the observed activation of antioxidant enzymes such as CAT and Se-GPx in the caudal kidney, as a defensive response to PFAS-induced oxidative stress. The complementary induction of these enzymes, varying across pollution levels, suggests a dynamic adjustment of the antioxidant system to the intensity of oxidative challenge.

In specimens sampled from the low-polluted site, there is a renal increase in Se-GPx activity but not in CAT. The opposite occurs in the medium-polluted site. Notably, antioxidant defences work in a complementary manner: Godin and Garnett (1992) previously correlated a low GPx activity with a high CAT activity; the opposite condition was pointed out in the liver of two Antarctic fish species, where high Se-GPx activity corresponded to lower CAT activity (Santovito et al., 2012). CAT typically plays a minor role at low H₂O₂ concentrations but becomes useful when this reactive oxygen species (ROS) increases (Regoli et al., 2005). Therefore, we can assume that Se-GPx has a first-line defence role against PFAS-induced oxidative stress, while CAT activity is enhanced when the risk of oxidative stress increases. In the highly-polluted site, both enzymes’ high activities were expected as ROS production could be so high that all antioxidant defences are required to counteract oxidative stress. The cooperation of different antioxidant system components is crucial, especially if the studied fish come from an environment subjected to viability in PFAS concentrations over time, so they must be ready to counteract future PFAS damages effectively.

The expressions of cat, gpx-1, and gpx-4 mRNAs were measured in the liver and caudal kidney and compared among the four sampling sites, whether polluted or not by PFAS, and with the amount of the relative active proteins for each site.

Another interesting feature of the activation of the antioxidant system in S. cephalus exposed to PFAS is the mismatch between the expression profiles at the gene (mRNA) and protein (enzyme activities) levels, which is not directly attributable to the different half-lives of the respective molecules. The absence of correlation between gene transcription and messenger translation referring to anti-stress proteins, such as those of the antioxidant system, was documented in our previous works in aquatic organisms (Ferro et al., 2013; Drago et al., 2022), including fish (Sattin et al., 2015; Tolomeo et al., 2019; Bakiu et al., 2022b; Pacchini et al., 2023). Many authors attribute this phenomenon to a post-transcriptional control on protein synthesis operated by stress granules (SGs) (Takahashi et al., 2013; Harvey et al., 2017; Curdy et al., 2021; Drago et al., 2021; Bakiu et al., 2024). These non-membranous cytoplasmic foci operate in the recruitment of silenced mRNAs due to the presence of specific mRNA-binding proteins (Drago et al., 2021; 2023; Piva et al., 2024). The mechanism by which these proteins operate in mRNA selection within SGs remains a subject of study (Khong et al., 2017; Glauninger et al., 2024). However, SG-based regulation could explain why increased CAT and Se-GPx activity in the caudal kidney did not match increased mRNA expression for the cat and gpx-1 genes. Indeed, the presence of PFAS-induced oxidative stress could induce the disassembly of SGs, leading to the release of specific mRNAs contained therein, which unlocks their translation and facilitates the biosynthesis of antioxidant enzymes.

The potential interaction between SGs and the antioxidant system requires future confirmation, and investigations into this effect are already underway. What is already evident is that not all components of the antioxidant system are subject to such regulation. Indeed, in the caudal kidney, our data indicate that the gpx-4 expression profile perfectly correlates with Se-GPx activity, thus suggesting that this isoform makes the main contribution to enzyme formation. Like other GPx isoforms, GPx-1 is predominantly expressed inside the cytoplasm, whereas GPx-4 is the only one present inside mitochondria (Pei et al., 2023), which are considered one of the PFAS intracellular toxicity targets (Hagenaars et al., 2013). Therefore, GPx-1 is probably not the most essential isoform in response to the stress caused by these pollutants.

The results obtained in this study provided valuable insights into the physiological strategies adopted by S. cephalus to survive in PFAS-contaminated rivers in the Veneto region, highlighting its potential as a model organism for assessing the impact of PFAS on European freshwater fauna. These strategies involve activating the antioxidant system in the caudal kidney, particularly Se-GPx and CAT, and the accumulation of lipids in the liver and caudal kidney to reduce the reactivity of PFAS to their target molecules, i.e., proteins. This study highlights the importance of monitoring the riverine fauna in its ability to acclimatise to an environment where human impact is becoming increasingly important.

There is certainly still much to evaluate and improve, such as the analysis of PFAS accumulation in different organs and tissues, which remains hindered by numerous methodological limitations.

Future research will expand the investigation of PFAS accumulation and anti-stress responses to a broader range of organs, including the brain, in addition to the kidney and liver analysed in the present study, despite the persistent methodological challenges in accurately quantifying tissue PFAS concentrations. The effects of PFASs on the brain are little known in fish. Still, it appears that they may alter brain function by interfering with amino acid neurotransmitter metabolism and disrupting blood-brain barriers (Wang R. et al., 2023), thus influencing the behaviour and inducing stress in these animals, similarly to other environmental perturbations (Schumann et al., 2023; 2025). Particular focus will be placed on histological confirmation of the inflammatory condition in the spleen, aiming to shed light on the development of specific pathologies resulting from chronic stress induced by PFAS, which may be a consequence of constant ROS production (Lushchak, 2016). In this respect, it will also be necessary to evaluate the role played by other components of the antioxidant system that are often overlooked, such as the aforementioned Prdxs or methionine sulphoxide reductase, which plays a key role in repairing oxidative damage (Ricci et al., 2017). And of course, the oxidation levels of lipids and proteins to better understand whether this new steady state of ROS is actually kept under control by the antioxidant system of S. cephalus (Piva et al., 2022) and, therefore, whether these fish will be able to survive a possible future increase in the environmental concentration of PFAS.