Hunting Metabolic Biomarkers for Exposure to Per- and Polyfluoroalkyl Substances: A Review

Per- and polyfluoroalkyl substances (PFAS) represent a class of persistent synthetic chemicals extensively utilized across industrial and consumer sectors, raising substantial environmental and human health concerns. Epidemiological investigations have robustly linked PFAS exposure to a spectrum of adverse health outcomes. Altered metabolites stand as promising biomarkers, offering insights into the identification of specific environmental pollutants and their deleterious impacts on human health. However, elucidating metabolic alterations attributable to PFAS exposure and their ensuing health effects has remained challenging. In light of this, this review aims to elucidate potential biomarkers of PFAS exposure by presenting a comprehensive overview of recent metabolomics-based studies exploring PFAS toxicity. Details of PFAS types, sources, and human exposure patterns are provided. Furthermore, insights into PFAS-induced liver toxicity, reproductive and developmental toxicity, cardiovascular toxicity, glucose homeostasis disruption, kidney toxicity, and carcinogenesis are synthesized. Additionally, a thorough examination of studies utilizing metabolomics to delineate PFAS exposure and toxicity biomarkers across blood, liver, and urine specimens is presented. This review endeavors to advance our understanding of PFAS biomarkers regarding exposure and associated toxicological effects.


Introduction
Per-and polyfluoroalkyl substances (PFASs) encompass both perfluorinated and partially fluorinated alkyl compounds, characterized by the replacement or partial replacement of hydrogen atoms by fluorine atoms on all carbon atoms.These synthetic chemicals consist of a carbon backbone ranging from 4 to 18 carbon atoms in length, terminating with functional groups [1].Depending on the terminal functional groups, PFASs can be categorized into carboxylates, perfluorosulfonates, and phosphonates, among others.Furthermore, they can be classified based on carbon chain length, including perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA).Owing to their exceptional industrial properties, such as hydrophobicity, oleophobicity, heat resistance, high stability, and low surface tension, PFASs have been extensively utilized since the 1950s in various industrial and consumer applications.These applications include the production of polytetrafluoroethylene, leather goods, waterproof textiles, firefighting foams, shampoos, cosmetics, and food packaging materials [2,3].
Unfortunately, the widespread use of PFASs and their desirable properties in commercial products have also led to their pervasive detection in the environment [4][5][6].Currently, various concentrations of PFASs have been detected in environmental samples, animal serum, tissue samples, and human bodies worldwide, positioning PFASs as a new class of persistent organic pollutants, alongside polychlorinated biphenyls, organochlorine pesticides, and dioxins.Epidemiological and toxicological studies have shown that PFAS exposure results in toxic effects on the liver, nervous system, immune system, reproductive and developmental processes, genetics, and endocrine function, and may even induce tumors, although the metabolic toxicity remains unclear [7][8][9][10][11][12][13].
In recent years, representative PFASs such as perfluorooctane sulfonate (PFOS) have been listed under the Stockholm Convention [14].Although these regulatory interventions have significantly reduced the emissions of PFOS and PFOA, human exposure to these compounds persists due to their persistence and ongoing production in some developing countries [15].Moreover, the high-energy C-F covalent bond renders PFASs extremely resistant to degradation, leading to their persistence in the environment.They can contaminate water and food through direct discharge, migration, bioaccumulation, and biomagnification within marine ecosystems [16].Evidently, even at low exposure levels, the potential health hazards of PFASs cannot be overlooked.As the adverse effects of PFASs on the environment and human health become more widely recognized, and as they migrate and biomagnify through the food chain, PFASs have emerged as a significant environmental and health concern.
Metabolomics, the large-scale study of metabolites within organisms, tissues, and cells, is considered a promising tool for elucidating the associations between environmental pollutants and health, as well as the etiology of certain diseases [17].As an emerging technology, metabolomics characterizes small molecule metabolites present in cells and their roles in various biological processes, offering a novel and powerful approach to understanding the complex molecular responses induced by PFAS exposure.Despite extensive research on PFAS detection and toxicity, studies on the metabolic toxicity and biomarkers of PFASs remain limited.As metabolomics rapidly advances, reviewing the current state of research on PFAS metabolic toxicity and its biomarkers will provide timely guidance for future research in this crucial field.
This review briefly introduces the types of PFASs, sources of human exposure, and exposure characteristics.It summarizes the latest advancements in studying PFAS toxicity using metabolomics, reviews metabolic disturbances, potential exposure biomarkers, and effect biomarkers, and emphasizes metabolic toxicity and related metabolic biomarkers.

Introduction to PFAS
PFASs gained widespread application in consumer and industrial products during the 1990s due to their surfactant properties.Around the year 2000, the recognition of their potential toxicity led to the gradual phasing-out of legacy PFASs, primarily longchain perfluoroalkyl carboxylic acids or perfluoroalkyl sulfonic acids.Currently, industrial applications of PFASs are increasingly shifting towards short-chain PFASs or those with chlorinated carbon or ether bond structures, giving rise to emerging PFASs.

Classification
PFASs are classified based on their functional groups and molecular structures into carboxylic acids and sulfonic acids.The carbon chain length for carboxylic acids generally ranges from 6 to 18, while for sulfonic acids it typically ranges from 5 to 10. Due to restrictions on the use of traditional long-chain PFAS, several emerging substitutes for conventional PFASs have gradually appeared.These substitutes include short-chain compounds and those incorporating fluorinated chlorinated carbons or ether bonds.Consequently, long-chain PFCA and PFSA are referred to as "legacy PFAS", while the substitutes are termed "emerging PFAS".Common types of PFASs are listed in Table 1.

Sources
The widespread presence of PFASs in industrial emissions, untreated domestic wastewater, sewage treatment plants, and aqueous film-forming foams has led to their detection in the environment, food, and human bodies [18][19][20][21].The most significant pathways for human exposure to PFASs include food, drinking water, skin contact, indoor dust, and outdoor air, with food being the primary route [22,23].Consuming contaminated food and drinking water, such as vegetables, crops, fish, meat, or processed food affected by PFAS, as well as drinking PFAS-contaminated water, can lead to exposure [19,[24][25][26][27]. Additionally, contact with food packaging materials containing PFASs (e.g., food packaging paper and non-stick cookware) also poses a risk [28,29].PFASs in soil may indirectly harm human health through bioaccumulation in crops [26].
PFASs can transfer into the human body via the food chain, accumulating and magnifying at each trophic level [30].In plants, PFASs can be absorbed by roots from contaminated soil or water and then translocated to flowers, leaves, and fruits [31,32].Long-chain PFASs tend to remain in roots, whereas short-chain PFASs are more readily transported to fruits [32,33].Aquatic organisms ingest PFAS-contaminated sediments and seawater, with studies showing that short-chain PFASs easily accumulate in these organisms [34,35].In livestock, animals ingest PFASs through water and feed, which then accumulate in muscle and milk [36,37], subsequently entering the human food chain and increasing human exposure risk.

Human Exposure Pathway and Characteristics
Multiple PFAS compounds have been detected in human tissues such as blood, kidneys, brain, cerebrospinal fluid, liver, lungs, and placenta [38][39][40][41][42].This necessitates urgent research into PFAS exposure and its effects on these human samples.

Population Characteristics
Occupational groups such as firefighters using aqueous film-forming foam have higher PFAS exposure risk than the general population, where diet is a critical factor influencing PFAS exposure [43].High seafood consumption correlates with increased human PFAS levels [19].PFAS serum levels exhibit gender differences, generally higher in males than females [44,45].Menstruation and breastfeeding help reduce PFAS levels in females [46].PFASs can also be transmitted to infants through umbilical cord blood, placenta, and breast milk [47,48].Children are at higher risk of PFAS exposure than adults due to the presence of PFASs in formula milk and dairy products [27,49].

Distribution Trend
With the gradual elimination of legacy PFASs, their levels in human serum are declining [50].However, emerging PFASs can still be detected in serum, with some linked to adverse pregnancy outcomes and other toxic effects [51,52].

Absorption and Distribution
PFAS absorption and distribution in organisms are influenced by carbon chain length, half-life, species, and gender.Animal studies show that perfluorohexanesulfonic acid (PFHxS), perfluorobutanesulfonic acid (PFBS), PFOA, and PFOS are almost completely absorbed after oral administration [53].PFASs primarily enter the bloodstream through the intestinal barrier, binding to blood albumin and low-density lipoprotein, and then distribute to extra-intestinal organs [54].Due to the high affinity for liver fatty acid-binding proteins, PFAS accumulation in the liver is well-documented [55].PFASs can also undergo enterohepatic circulation, re-entering the intestines via bile and returning to the liver, leading to high liver concentrations [56].Generally, longer carbon chains correspond to longer half-lives, ranging from hours to decades [57,58].The half-life also varies by gender and species [59,60].Current research mainly focuses on PFCA and PFSA, with the absorption, distribution, toxicokinetics, and metabolic toxicity of emerging PFASs requiring further investigation.

Toxic Effects of PFAS
Existing research has demonstrated that PFASs exhibit a range of toxic effects, including thyroid toxicity, hepatotoxicity, immunotoxicity, endocrine disruption, neurotoxicity, reproductive and developmental toxicity, and even carcinogenic and cancer-promoting properties.Additionally, elevated cholesterol levels, obesity, and endocrine disorders are associated with PFAS exposure.Although the connection between PFASs and toxicity in specific tissues and organs is recognized, the mechanisms through which PFASs influence metabolism and subsequently affect organ toxicity remain to be fully elucidated.This review summarizes the epidemiological and rodent model evidence linking PFAS-induced liver injury, reproductive and developmental toxicity, cardiovascular diseases, renal toxicity, and cancer, with a focus on metabolomics insights to better understand the metabolic mechanisms underlying PFAS exposure and toxic effects.
Metabolomic studies have revealed that metabolic disturbances are critical in PFASinduced liver damage.PFASs disrupt hepatic metabolism, particularly bile acid, amino acid, and lipid metabolism, leading to liver injury or influencing the development and susceptibility to NAFLD and liver damage [62,63,69,70].Sen et al., through a correlation analysis of PFAS (PFOS, PFNA, PFOA) exposure levels with metabolic perturbations in the liver in 105 individuals with NAFLD, found that PFASs perturbed key metabolic pathways of bile acid metabolism and lipid metabolism in NAFLD, and that there were gender differences [63].Similarly, it has been noted that high PFAS exposure levels were associated with more severe histology of liver damage in children with NAFLD, which may be related to elevated plasma levels of phosphoethanolamine, tyrosine, phenylalanine, aspartic acid, and creatine, and altered metabolites with reduced betaine levels [62].Additionally, it has been found that prenatal PFAS exposure increases susceptibility to liver damage in childhood, associated with branched-chain amino acids, aromatic amino acids, and glycerophospholipid metabolism [69].In addition, PFAS mixtures were found to be positively correlated with polipoprotein B (APOB) and γ-glutamyltransferase (GGT), and negatively correlated with direct bilirubin (DBIL) and total bilirubin (TBIL) [70].
Researchers analyzing metabolite changes in the liver or blood after PFAS exposure in rodents also show that PFASs affect bile acids, sterols, fatty acids, purine, and amino acid metabolism, contributing to liver injury [66,68,[71][72][73].For example, exposure to PFAS mixtures leads to liver injury in mice, such as increased liver weight, inflammation, and elevated plasma ALT levels, with significant changes in bile acid and sterol metabolism [71].Lipidomic studies suggest that PFOS disrupts ceramide and lysophosphatidylcholine (LPC) metabolism, causing apoptosis and triglyceride depletion in liver cells, leading to morphological liver damage [72].Functional characterization of lipid species showed that the fatty acid metabolic pattern of A/J mice was significantly altered by PFAS mixture exposure, especially the biosynthetic pathways of glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and glycerophosphoserine (PS) in the liver were significantly affected [73].In addition to bile acids, sterol, fatty acid, amino acid, and purine metabolism were also affected [66,68].Jiang et al. examined liver nontargeted metabolomics after PFHxA exposure in mice and found that PFHxA substantially downregulated xanthine and uric acid, increased GSH, and decreased GSSH, which presumably caused oxidative stress by interfering with purine metabolism and glutathione metabolism, thereby causing liver damage [66].Additionally, PFASs may influence liver damage by affecting gut microbiota metabolism, as evidenced by altered fecal microbiota and arginine metabolism in PFOS-exposed mice [68].

Reproductive and Developmental Toxicity
Studies indicate that PFASs adversely affect male reproductive function, including semen quality and male reproductive hormones [74].Similarly, PFASs impact female reproductive functions such as menstrual cycle regulation, hormone levels, and fertility [75].Furthermore, PFASs can cross the placental barrier, thereby influencing fetal development and contributing to adverse pregnancy outcomes like preterm birth [76], reduced birth weight [77,78], postnatal growth [79], and neurodevelopmental disorders [80,81].Additionally, PFAS exposure is linked to increased disease risks in offspring, such as liver damage susceptibility and type 1 diabetes [69,82].Research has identified a correlation between maternal PFAS co-exposure and decreased sperm concentration, with varying contributions from different PFASs, with perfluoroheptanoic acid as a primary factor.However, no clear association was found between maternal PFAS exposure and testicular volume or reproductive hormones [74].Contrarily, another study showed a significant negative correlation between PFAS mixtures and reproductive hormones, particularly estradiol (E2) and the E2/total testosterone (TT) ratio, with perfluoro-n-undecanoic acid (PFUdA) being a major contributor [83].Higher serum concentrations of PFOA and PFHxS in pregnant women are associated with increased odds of preterm birth [76].PFASs also adversely affect prenatal and postnatal neurodevelopment.Additionally, prenatal PFAS exposure is linked to neurodevelopmental disorders in children, such as reduced IQ performance and impaired executive function [80].
Population studies using metabolomics found that the effects of PFASs on preterm birth, fetal growth, childhood obesity trajectory, susceptibility to liver injury, and other reproductive developmental processes were related to amino acid, glycerophospholipid lipid and fatty acid, bile acid, uric acid, and carbohydrate metabolism [69,76,79,84,85].Two studies found that PFAS exposure was associated with preterm delivery or fetal growth in pregnant women and that the effects were associated with metabolite alterations, e.g., one study found that the impact of prenatal PFAS exposure on the shortening of the gestation period was associated with eight metabolomic pathways and 52 metabolites in the neonatal blood spot [76,84].Another study also found that higher concentrations of PFOA and PFNA in maternal serum were associated with reduced fetal growth and were strongly associated with disturbances in amino acid, lipid and fatty acid, bile acid, and androgen metabolism [84].Two other studies found that prenatal exposure to PFASs was associated with increased susceptibility to liver damage and an obesity trajectory in children, as well as with metabolites such as amino acids, glycerophospholipids, sphingolipids, and octanoylcarnitine [69,79].Consistent with human studies, laboratory research on rodents has demonstrated that the effects of PFASs on reproductive development were associated with metabolite disruption.For instance, pregnant Sprague-Dawley rats exposed to nafion byproduct 2 (NBP2), a type of perfluoroalkyl ether sulfonate, experienced neonatal mortality, reduced pup weight, and decreased liver glycogen in pups, as well as significant alterations in lipid and carbohydrate metabolism in dams and offspring [85].

Cardiovascular Toxicity
Cardiac metabolism, including conditions such as obesity, hypertension, and hyperlipidemia, has been extensively studied in relation to PFAS exposure.This review focuses on lipid and cholesterol abnormalities.Research indicates that twelve PFAS-related characteristic metabolites are associated with cardiovascular diseases [86].A study examining the relationship between Cl-PFESA, a typical PFAS substitute in China, and the lipid profiles of 1336 residents in Guangzhou revealed that increased Cl-PFESA levels were positively associated with total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels.The significant positive correlation between Cl-PFESA and lipid abnormalities suggests a detrimental effect of Cl-PFESA on lipid profiles, potentially exhibiting a nonlinear association [87].
Metabolomic studies have shown that PFASs primarily cause lipid and cholesterol abnormalities by disrupting bile acid and sterol metabolism [71,[88][89][90].Two population-based studies using metabolomics have found that PFASs were associated with plasma triglycerides and that this association was related to metabolite alterations [88,89].Another study revealed that PFAS exposure was linked to a higher risk of dyslipidemia, and associations between PFASs and TC and LDL were found to be primarily related to glycerophospholipid metabolism, primary bile acid biosynthesis, and linoleic acid metabolism [90].Animal studies have also demonstrated that PFASs interfere with metabolism, leading to increased circulating cholesterol and cholesterol metabolism abnormalities.For instance, C57BL/6J mice fed a high-fat diet and exposed to a PFAS mixture exhibited elevated fasting plasma cholesterol levels [71].The levels of several sterol metabolites, including 4-cholesten-3one, were upregulated, while most primary bile acids, such as chenodeoxycholic acid and β-muricholate, were significantly reduced in the liver metabolome.These findings suggest that PFASs may disrupt cholesterol metabolism and transport, contributing to a PFAS-induced cholesterol imbalance.

Glucose Homeostasis Disruption
PFASs are closely associated with endocrine disorders such as diabetes, impaired glucose tolerance, overweightness, or obesity [91].Epidemiologic studies have found that PFOA and PFOS exposure were associated with higher 30 min blood glucose levels and area under the glucose curves (AUCs) during oral glucose tolerance tests (OGTTs), as well as higher levels of HbA1c [89,92].Additionally, research indicates that PFOA and PFHxS were associated with increased 2 h glucose levels and increased area under the glucose curve, as well as altered lipid (e.g., sphingolipids, linoleic acid, and de novo lipogenesis) and amino acid (e.g., aspartic acid and aspartate, tyrosine, arginine, and proline) metabolism [93].Laboratory evidence further suggests that gastric gavage of pregnant rats with low doses of PFOSs disrupts critical metabolic products, such as glycerol-3-phosphate and lactosylceramide, affecting fasting blood glucose (FBG) in mothers and thus impacting glucose homeostasis [94].

Other Toxicological Impact
Studies have demonstrated that PFASs can also interfere with metabolism and impact the development of kidney diseases and cancer [95][96][97].Research indicates a causal relationship between PFASs and declining kidney function and chronic kidney disease [95].A study identified eight potential biomarkers associated with PFAS exposure, all significantly correlated with kidney function markers, suggesting adverse effects of high PFAS exposure on kidney function [96].Furthermore, PFASs have been implicated in liver cancer risk, possibly through metabolic interference.For instance, plasma PFAS concentrations are positively correlated with hepatocellular carcinoma (HCC) risk, with PFOSs showing the strongest correlation, associated with increased HCC incidence [97].Metabolomic analysis revealed 433 metabolites in plasma associated with PFOSs and 499 metabolites associated with HCC.

Potential Biomarkers
The metabolome encompasses the complete set of small molecules that participate in physiological processes within the body or in cells and tissues.The application of high-throughput analytical techniques has emerged as a novel tool for the comprehensive examination of endogenous metabolites in vivo.Research has demonstrated that pollutants can disrupt metabolic pathways, leading to alterations in metabolite levels within the human body.These changes in metabolites may indicate defects in specific pathways or the activation of certain signals, and the dynamic alterations of these metabolites can serve as biomarkers of pollutant-induced damage to the human body.Metabolomics can reveal toxicological changes and related mechanisms at an earlier stage and can be utilized as a tool for discovering biomarkers of pollutant exposure and effects.Metabolomics primarily focuses on the quantitative analysis of metabolites under specific physiological conditions following organismal exposure, understanding metabolic changes under varying conditions, and elucidating the associations between these changes and the organism's health status and disease.Therefore, comprehending the relationship between PFAS exposure and metabolites within the body can provide additional evidence for elucidating the health risks posed by pollutant exposure.

Exposure Biomarkers
Metabolomics methodologies emphasize the analysis of primary and secondary metabolites, which are crucial for obtaining the phenotypic fingerprints of organisms in their environments.These metabolic gatekeepers can also elucidate molecular connections between exposure biomarkers and health outcomes, which might otherwise be obscured by the complex interactions present in direct measurements [98].By focusing on the quantitative analysis of metabolites in organisms exposed to PFASs under specific physiological conditions, metabolomics can identify metabolic changes induced by PFASs and their associations with health status.Consequently, we concentrate on the quantitative analysis of metabolites in blood, liver, and urine under specific physiological states of PFAS exposure, summarizing the disturbances in metabolites in both human populations and rodent models post-PFAS exposure to identify potential PFAS-related exposure biomarkers.
Current research on PFAS exposure and metabolic alterations primarily involves human and rodent subjects.The studies cover various PFAS compounds, with the most extensive research on PFOA, PFOS, PFHxS, PFNA, and PFDA.There are also some metabolomics studies on emerging PFASs, such as 6:2 FST, 6:2 Cl-PFESA, GenX, NBP2, and FTEOs, though most research focuses on the effects of individual PFAS compounds, with some addressing the impact of mixtures.In the study of PFAS-related metabolites, alterations in amino acids (with branched-chain amino acids being more prominently affected), lipids, bile acid metabolism, and the urea cycle are common metabolomic features.Glycerophospholipid metabolism in lipid pathways is considered a key metabolic feature, along with fatty acid and carnitine metabolism related to fatty acid oxidation and energy supply pathways.Purine and pyrimidine metabolism in cellular energy systems are also identified as significant metabolic changes, with potential as future exposure biomarkers [99].
Most current studies utilize metabolomics in the blood (serum, plasma, cord blood) and liver to identify related metabolites, with fewer studies on urine and placental metabolite changes.Some studies simultaneously use serum or plasma and liver samples to explore the impact on enterohepatic circulation.Given the greater practicality of detecting metabolites in different samples for clinical translation, we summarize recent studies and advancements in metabolomics from various samples, as detailed in Tables 2 and 3.

Blood
As illustrated in Tables 2 and 3, in recent years there have been 19 studies investigating the effects of PFASs on metabolomic alterations in blood (plasma, serum, or cord blood).In human studies, PFAS exposure detection and metabolomic analyses typically utilize the same sample matrix, encompassing the general population, special populations (pregnant women, fetuses, children), occupational groups, and diseased cohorts.Diseased cohorts primarily include overweight or obese children, adolescents or young adults, adults and children with NAFLD, HCC cases, and individuals at high risk for or diagnosed with type 2 diabetes.Additionally, a few studies have inconsistent samples for PFAS exposure detection and metabolomic analyses, such as examining maternal PFAS exposure and subsequent metabolomic changes in cord blood, newborn, or child blood to investigate prenatal exposure effects on offspring blood metabolomics.
The close association of PFAS exposure with the metabolism of lipids, amino acids, and bile acids is also well documented in metabolome-related studies in disease populations [62,93,97,105].In studies of PFAS-related metabolites examined in children with NAFLD, in populations at high risk for diabetes, and in overweight and obese populations, it was found that amino acid and glycerophospholipid metabolisms were most relevant after PFAS exposure [62,93,105].For example, the pathways found to be most affected after PFAS exposure in children with NAFLD include tyrosine metabolism, alanine and aspartate metabolism, glycine, serine, alanine and threonine metabolism, urea cycle metabolism, and glycerophospholipid metabolism [62].Differential metabolites have been found in overweight and obese children, mainly associated with amino acids such as aspartate, tyrosine, arginine, and proline and lipid metabolic pathways such as sphingolipid metabolism, fatty acid metabolism, de novo lipogenesis, and linoleic acid metabolism [93].In people at high risk of diabetes, PFASs are mainly associated with amino acid and glycerophospholipid metabolism, and PFASs are closely related to branched-chain amino acids (isoleucine, leucine, and valine) [105].In addition, metabolites have been found to be closely associated with PFOS in HCC cases, mainly interfering with amino acids, carbohydrates, and glycan biosynthesis, and metabolism in relation to them [97].

Liver
The impact of PFAS exposure on liver metabolites primarily manifests in disruptions to bile acid and lipid metabolism, as well as effects on amino acid, purine, and glutathione metabolism.Human studies on liver metabolomics are limited, likely due to the difficulty in obtaining liver samples.However, one study involving liver biopsies from individuals with NAFLD undergoing laparoscopic surgery analyzed the relationship between PFAS exposure levels in blood and liver metabolites, finding that PFAS exposure was primarily associated with disruptions in liver bile acid and lipid metabolic pathways [63].In individuals with NAFLD, studies have demonstrated gender-specific associations between PFAS exposure and liver metabolites.In females, serum concentrations of PFASs (PFNA, PFOA, and PFOS) positively correlated with primary liver bile acids (TCA, GCDCA, TCDCA), while PFOA was positively associated with various secondary bile acids (DCA, GHCA, and GUDCA).Additionally, PFOA and PFOS were positively correlated with ce-ramides (e.g., Cer(d18:0/16:0), HexCer(d18:1/18:0)), ether phospholipids (e.g., O-PC(40:4)), TGs), and diglycerides (DGs).These associations were not observed in males.For both sexes, shared metabolic impacts included overexpression of primary bile acid biosynthesis, glycerophospholipid metabolism, and alanine, aspartate, and glutamate metabolism.Similar lipid associations were observed in mice, where PFOA exposure was linked to Cer, hexosylceramides(HexCer), LPE, DG, and TG.Most studies linking PFAS exposure and liver metabolomics focus on rodent models, particularly targeting lipid metabolism.Three animal studies have all found that PFAS exposure leads to alterations in the hepatic metabolome and is primarily associated with lipid metabolism [72,73,106].For example, Kirkwood-Donelson KI et al. studied the effects of NBP2 or GenX exposure on the hepatic metabolome and found that over half of the detected lipids were significantly dysregulated, with oleic acid (OA) and dihomo-γ-linoleic acid (DGLA) being the most commonly affected fatty acids [106].Khan et al. found that, similar to the blood metabolome, lipid differential metabolites were dominated by eight different lipid major classes, including Cer, DG, PC, PE, PS, SM, sterols, and TG, with common lipid species across genders including PC 38:4, PC 38:5, PC 38:6, TG 52:5, TG 56:5, and PE 38:6 [73].In addition, further studies on fatty acids revealed that four fatty acids, alpha-linolenic acid, DHA, DPA, and FA 20:4, were significantly different between males and females.Li et al. analyzed PFAS exposure and lipid metabolites and found that 54 lipids had differentiated lipid metabolites and that the major abnormal lipids were glycerophospholipids, sphingolipids, and TG [72].In addition, in studies analyzing the metabolism of sterols, ketone bodies, and acylcarnitines in the liver, it was found that sterol metabolites in the liver mainly showed a decrease, ketone bodies an increase, and most of the acylcarnitines an increase [71].In addition, two animal studies have found that PFAS exposure is associated with amino acid, purine, and glutathione metabolism [66,94].Among them, Yu et al. found that more metabolites were found in the negative ion pattern and more differential metabolites were up-regulated after PFAS exposure.These metabolites were enriched in pathways such as α-linolenic acid metabolism, linoleic acid metabolism, purine metabolism, glycine, serine, and threonine metabolism, steroid biosynthesis, glycolysis/gluconeogenesis, and glucagon signaling pathways [94].Jiang et al. found that PFHxA exposure was also associated with purine and glutathione metabolism [66].

Urine and Placenta
Limited research has examined metabolite changes in urine and placenta.He et al. found that PFAS concentrations in urine closely correlated with those in serum among workers and residents near a large fluorochemical plant in Hubei Province, China, suggesting that urinary PFAS could serve as a good indicator of serum PFAS levels [96].Eight potential exposure biomarkers were identified, with differential metabolites associated with amino acids, steroids, fatty acid derivatives, carboxylic acids, isoprenoids, amines, and vitamin pathways.Adams et al. investigated placental metabolites in healthy CD-1 pregnant mice exposed to PFOA and FTEOs, identifying significant changes primarily in amino acids [107].Fourteen different metabolites, including asparagine, fatty acids, glucose, threonine, lactate, lysine, and creatine, were identified.PFOA exposure led to increased glucose and threonine and decreased creatine in the placenta, whereas FTEO exposure decreased asparagine and lysine while increasing creatine.Pathway analysis indicated alterations in glycine, serine, and threonine metabolism, as well as valine, leucine, and isoleucine biosynthesis following PFOA exposure, with biotin metabolism affected by FTEO exposure.

Effect Biomarkers
There is a substantial body of research on the relationship between PFAS exposure and metabolites; however, studies that connect these metabolites to both PFAS exposure and biological effect indicators are limited, primarily focusing on changes in blood metabolites.To date, metabolomics research has enhanced our understanding of the comprehensive impact of PFAS exposure on human health and has identified associated metabolites and intermediary markers involved in the onset and progression of various diseases (e.g., liver diseases, glucose and lipid abnormalities, cancer).These metabolites may serve as potential biomarkers for the association between PFAS exposure and biological effects.By synthesizing recent metabolomic studies on PFAS exposure and toxicity, we can elucidate consistent metabolic response patterns to PFAS exposure, revealing common pathways and potential biomarkers affected in both human models and laboratory rodents, as detailed in Table 4.

Blood
Research on the association between PFASs and liver and glucose-lipid abnormalities has identified glycerophospholipids as major associated metabolites with potential as effect biomarkers.Four studies have all found that glycerophospholipids may be the mediating metabolite that mediates the association of PFAS exposure with liver function or dyslipidemia [62,70,88,90].A mediator analysis of metabolites mediating the association of PFAS exposure with abnormalities in APOB, an index of liver function, showed that glycerophospholipids were the main markers for the association of PFASs with APOB [70].PFASs were also found to be predominantly associated with glycerophospholipid and amino acid metabolism in a study of the histologic severity of nonalcoholic fatty liver disease, in which differential metabolites were divided into two potential clusters, and cluster 2 was found to be associated with an increased incidence of NASH and higher PFAS concentrations [62].In dyslipidemia studies, most glycerophospholipids were positively associated with PFASs and were associated with an increased risk of TC abnormalities, and the metabolites that played a mediating role mainly involved glycerophospholipid metabolism, linoleic acid metabolism, and primitive bile acid biosynthesis, in which 24-Hydroxycholesterol, 3alpha,7alpha-dihydroxy-5beta-cholestan-26-al, PC(18:0/0:0), PC(22:5/0:0), GPCho(18:1/18:1), LPC(22:2(13Z,16Z)), LPC(16:0), 9(S)-HODE, 9,10-DHOME, L-glutamate, 4-hydroxybutyric acid, cytosine, PC(14:1(9Z)/18:0), sphinganine, and (S)-beta-aminoisobutyrate were important markers [90].Similarly, in studies related to plasma cholesterol and triglycerides, a metabolite pattern predominantly involving glycerophospholipids mediated the association between PFASs and triglycerides, showing a negative correlation with triglycerides [88].In HCC risk studies, high PFOS levels were associated with increased HCC risk, with glucose, butyric acid, α-ketoisovaleric acid, and 7α-hydroxy-3-oxo-4-cholestenoate identified as potential mediators of the association between PFOS exposure and increased HCC risk [97].The possible mechanism linking PFASs to increased liver cancer risk is through alterations in glucose, amino acid, and bile acid metabolism.In type 2 diabetes (T2D) risk studies, PFASs were found to be positively associated with two metabolite patterns, but these patterns had opposite relationships with T2D risk [91].The metabolite pattern PC2, predominantly involving glycerophospholipids, was associated with reduced T2D risk, whereas the metabolite pattern PC1, primarily involving diacylglycerols, was associated with increased T2D risk.In studies related to changes in glucose homeostasis, children with high PFAS concentrations were found to have elevated 2 h glucose levels at baseline and during follow-up [93].Further metabolomic analysis divided differential metabolites into two potential clusters, with the "high-risk" cluster positively correlated with PFASs, mainly involving the metabolism of palmitic acid, hydroperoxylinoleic acid, tyrosine, phenylalanine, arginine, sphingolipids, linoleic acid, and aspartic acid.
In the study of intermediary metabolites associated with PFASs and reproductive development, it was found that PFASs are primarily associated with the metabolism of amino acids and lipids and identified Octanoylcarnitine (C8) and uric acid as potential intermediary biomarkers [76,79,84].In studies examining maternal PFAS exposure and childhood obesity trajectories before age four, three BMI z-score trajectories in early childhood were identified, in which an increased likelihood of a persistently increasing trajectory type was associated with prenatal PFAS exposure [79].Further analysis identified octanoylcarnitine (C8) as the sole mediator between PFAS exposure and childhood BMI trajectory.In a study of maternal PFAS exposure associated with gestational age and PTB, PFASs and gestational age and PTB were found to be primarily involved in the pathways of glycerophospholipid metabolism, amino acid metabolism in the urea cycle, and tryptophan metabolism [76].In another study on PFAS exposure and fetal growth in pregnant women, 10 metabolites, such as glycine, taurine, uric acid, ferulic acid, and the unsaturated fatty acid C18:1, were identified as overlapping with the endpoints of PFASs and fetal growth.Further analysis indicated that uric acid may be a potential intermediate biomarker representing an early response to PFAS exposure and predicting reduced fetal growth [84].In addition, in studies on prenatal PFAS exposure and childhood liver injury risk, an increase in prenatal PFAS exposure was associated with elevated serum levels of branched-chain amino acids (BCAAs: valine, leucine, and isoleucine), aromatic amino acids (AAAs: tryptophan and phenylalanine), biogenic amine acetylornithine, glycerophospholipid PC aa C36:1, and Lyso-PC a C18:1 in children at high risk for liver injury compared to those at low risk [69].

Summary
Exposure to PFASs has been associated with various biological abnormalities.The advancement of metabolomics technology has provided us with tools to develop an earlier and more comprehensive understanding of the impact of PFAS exposure on disease occurrence and progression mechanisms, as well as facilitating the identification of exposure and effect biomarkers.As illustrated in Figure 1, existing research indicates that PFASs primarily affect liver damage, reproductive toxicity, cardiovascular diseases, abnormal glucose metabolism, kidney diseases, and cancer by influencing the metabolism of amino acids, lipids, and bile acids.Several animal experiments have demonstrated a dose-response relationship of PFASs to alterations in lipid and amino acid metabolites, such as in the metabolome of 0.03 and 0.3 mg/kg PFOS-exposed pregnant rats, where a dose-response relationship was also found in bile secretion, glycine, serine and threonine metabolism, and linoleic acid metabolism, as well as a dose-response relationship in the liver transcriptome in terms of alterations in bile secretion, valine, leucine and isoleucine biosynthesis, and arachidonic acid metabolism [94].Gao et al. observed an effect of PFOA on lipids, such as unsaturated triglycerides, sphingomyelins, saturated phosphatidylcholines, phospholipid ethers, and other lipids, in a dose-response relationship [108].In addition, reports of BA metabolism in animal models and in vivo studies have demonstrated that PFAS exposure inhibits de novo synthesis of BA via inhibition of cholesterol 7a-hydroxylase (CYP7A1) and that subsequent inhibition of CYP7A1 leads to the down-regulation of primary BAs (CA, CDCA) [63,71].Current studies suggest that PFASs primarily induce liver damage by disrupting bile acid, amino acid, and lipid metabolism, reproductive toxicity by perturbing amino acid, lipid, fatty acid, glycerophospholipid, bile acid, uric acid, and carbohydrate metabolism, and lipid and cholesterol abnormalities by disturbing bile acid and cholesterol metabolism.Additionally, PFASs can also influence the occurrence and development of glucose metabolism, kidney diseases, and cancer through metabolic regulation.Although the specific metabolites disrupted by toxicity in different tissues and organs may vary, there are common metabolic disturbances, such as associations with amino acids, lipids, and bile acids observed in almost all abnormalities.Changes in various metabolites in blood, liver, and urine were associated with exposure to PFASs, which offers a reservoir of metabolites that may be associated with PFAS exposure.Some unique metabolites or a combination of multiple metabolites could be potential biomarkers upon further validation.Reviewing the past literature reveals that metabolites associated with PFASs primarily include lipids, amino acids, bile acids, steroids, and acylcarnitines, with changes in blood metabolites dominating the research.Glycerophospholipids show promising potential as effect biomarkers in studies related to PFAS exposure and toxic effects.In addition, we have some limitations with this review in that the alterations in metabolites such as lipids, amino acids, and bile acids identified by this study may not be unique to PFASs alone; however, a comprehensive characterization of the metabolite changes would provide clues to the underlying mechanistic alterations that underlie these adverse outcomes.Further research is needed for a more specific investigation into lipid and amino acid metabolism.Moreover, due to differences in sample matrix and physiological pathological states, different biomarkers may be identified, emphasizing the need for further analysis of specific biomarkers under specific physiological conditions.

Outlook
Current research on PFAS exposure primarily focuses on legacy PFAS compounds, with a predominant emphasis on individual PFAS studies.Some studies have indicated that disturbances in metabolomic profiles caused by PFAS mixtures may be more pronounced than those induced by single PFAS compounds.However, the composite exposure characteristics of perfluoroalkyl substances and their effects on internal metabolites remain unclear.This significantly hampers the accuracy of risk assessments for populations with high exposure to perfluoroalkyl substances.Therefore, there is a need to fill the data gap in metabolomic studies of novel PFAS substitutes and PFAS mixtures.It has also been observed that the toxicity varies among different monomers.Even with higher accumulation levels in the liver, the disruptive effects of their

Outlook
Current research on PFAS exposure primarily focuses on legacy PFAS compounds, with a predominant emphasis on individual PFAS studies.Some studies have indicated that disturbances in metabolomic profiles caused by PFAS mixtures may be more pronounced than those induced by single PFAS compounds.However, the composite exposure characteristics of perfluoroalkyl substances and their effects on internal metabolites remain unclear.This significantly hampers the accuracy of risk assessments for populations with high exposure to perfluoroalkyl substances.Therefore, there is a need to fill the data gap in metabolomic studies of novel PFAS substitutes and PFAS mixtures.It has also been observed that the toxicity varies among different monomers.Even with higher accumulation levels in the liver, the disruptive effects of their metabolites may not necessarily be greater [85,106].Studies have highlighted that the novel PFAS substitute NBP2 exhibits developmental toxicity in rats, with oral toxicity slightly lower than PFOS but stronger than HFPO-DA.Comparing the toxicity of different PFAS monomers is crucial for identifying substitutes with lower toxicity.Thus, further research is needed to compare the toxicity of different novel PFAS compounds.
Existing research on the association between PFAS exposure and metabolomics mainly focuses on changes in metabolites associated with PFAS exposure to explore underlying mechanisms.However, there is a lack of research on exposure biomarkers and metabolomic markers that further mediate these effects.
Research on the biological effects of PFASs primarily targets the general population, with limited studies on the effects of exposure on pregnant women, infants, toddlers, and children.Additionally, studies have found higher detection rates among occupational populations near factories, with differential metabolites associated with oxidative stress, impaired fatty acid β-oxidation, and kidney damage.The impact of occupational exposure on worker health cannot be overlooked, highlighting the need for further research on populations with high exposure risks [100].
With the advancement of omics technologies, transcriptomics, metagenomics, and proteomics have become valuable tools for studying the biological effects of PFAS exposure.However, current research lacks comprehensive multi-level investigations combining metabolomics with other omics approaches to elucidate the mechanisms underlying the occurrence and development of PFAS-related effects.There is a need to integrate various omics techniques to provide a more comprehensive understanding of the impact of PFAS exposure and to validate metabolomics findings across multiple levels using other omics methodologies.

Table 2 .
PFAS exposure and associated metabolites in epidemiological studies.

Table 3 .
PFAS exposure and associated metabolites in rodent studies.

Table 4 .
PFAS exposure intermediate metabolites associated with biological effects.