Comparative study on changes in serum amino acid metabolic profiles in mice exposed to perfluorooctanoic acid and sodium ρ-perfluorous nonenoxybenzene sulfonate

Abstract

Perfluorinated compounds (PFCs) such as perfluorooctanoic acid (PFOA) and sodium ρ-perfluorous nonenoxybenzene sulfonate (OBS) are toxic to organisms, but their toxicological differences in affecting serum amino acid metabolism remain unclear. To investigate the effects of subchronic PFOA and OBS exposure on mice’s serum amino acid metabolic profile and explore their toxicological differences. Fifteen healthy male C57BL/6N mice were randomly divided into three groups: control (CON) group, PFOA group (exposed to 3 mg/(kg·d) PFOA), and OBS group (exposed to 3 mg/(kg·d) OBS). The experiment was conducted continuously for four weeks. After the exposure period, serum samples were collected, and the concentrations of free amino acids and their derivatives in the serum were determined using an automatic amino acid analyzer. Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used for data analysis. PCA and OPLS-DA results showed distinct intra-group clustering and clear inter-group separation of the serum amino acid metabolic profiles. A total of 23 differential amino acids and their derivatives were identified with the criterion of variable importance in projection (VIP) > 1.0. Quantitative analysis indicated that neither PFOA nor OBS exposure significantly altered the total serum amino acid levels in mice, but both selectively disrupted the metabolic homeostasis of specific amino acids and their derivatives. For essential amino acids, exposure to either PFOA or OBS significantly increased serum leucine levels; serum threonine levels were significantly decreased in the PFOA group but increased in the OBS group. For non-essential amino acids, the levels of serine, asparagine, and glutamine in the OBS group were significantly higher than those in both the CON and PFOA groups. Regarding amino acid derivatives, 11 compounds showed highly significant differences (P < 0.01) and 3 showed significant differences (P < 0.05) among the three groups. Subchronic exposure to both PFOA and OBS significantly disrupts the serum amino acid metabolic profile in mice. However, the two compounds exhibited distinct effects: OBS exerted a broader impact on non-essential amino acids (serine, asparagine, glutamine), while PFOA showed a stronger association with cardiovascular and muscle toxicity markers (homocysteine, 1-methylhistidine). These findings suggest that PFOA and OBS may exert toxic effects through different molecular mechanisms.

Introduction

Perfluorinated compounds (PFCs) are a class of synthetic organic pollutants with unique physicochemical properties. Owing to their excellent heat resistance, corrosion resistance, and surface activity, they have been widely applied in textiles, chemical industry, food packaging, and other fields^1,2. As a typical member of PFCs, perfluorooctanoic acid (PFOA) is recalcitrant to degradation in the environment due to the extremely high bond energy of its carbon-fluorine (C-F) bonds³. It can accumulate in organisms through multiple pathways such as the food chain and drinking water, ultimately posing potential threats to the ecological environment and human health^4,5. Consequently, PFOA has been recognized as a globally concerned pollutant. Previous studies have demonstrated that PFOA exposure significantly alters the amino acid metabolic profiles in the brains and livers of mice, including central nervous system-related amino acids (e.g., glutamic acid, γ-aminobutyric acid) and key hepatic metabolic amino acids (e.g., branched-chain amino acids, aromatic amino acids). These findings suggest that PFOA may disrupt amino acid metabolic homeostasis, thereby exerting potential toxic effects on the neurological and hepatic functions of animals⁶. Additionally, PFOA exposure can disrupt the arginine metabolic pathway in the liver, ultimately leading to liver injury⁷. Sodium ρ-perfluorous nonenoxybenzene sulfonate (OBS) is a novel PFC with stronger surface activity and hydrophobicity. With the increasingly stringent restrictions on the use of traditional PFCs, the application of OBS as an alternative has gradually expanded in recent years⁸. Studies have indicated that subchronic environmental exposure to OBS can induce intestinal flora dysbiosis in animals and trigger hepatic metabolic disorders via the gut-liver axis. This process not only causes abnormalities in hepatic amino acid and lipid metabolic pathways but also activates oxidative stress and inflammatory pathways, thereby mediating liver injury⁹. However, few studies have focused on the effects of PFOA and OBS on amino acid metabolism in animal serum, and nor on their comparative analysis, which urgently requires further investigation.

Alterations in serum amino acid metabolic profiles can directly reflect the nutritional status, physiological functions, and pathological damage of organisms, and thus, serve as sensitive indicators for evaluating the toxic effects of exogenous chemicals^10,11. Amino acids are involved in a series of core physiological processes, including energy metabolism, protein synthesis, signal pathway regulation, and maintenance of immune function in organisms. The homeostasis of amino acid metabolism is therefore a crucial guarantee for the normal life activities of organisms. When exposed to exogenous pollutants, the amino acid metabolic pathways of organisms may be disrupted¹². Multiple studies have shown that the imbalance of amino acid metabolic homeostasis can trigger a series of health problems. For example, glutamine is excessively taken up and accumulated in tumor cells through highly expressed transporters on the tumor cell membrane. After decomposition by glutaminase, glutamine further abnormally activates the mTORC1 and MYC signaling pathways while disrupting the homeostasis of the tricarboxylic acid cycle, which leads to the dysregulation of tumor-related signaling pathways and an increased risk of the occurrence and progression of malignant tumors such as lung cancer and colorectal cancer¹³. In addition, phenylalanine impairs insulin signaling by modifying the insulin receptor β-subunit (IRβ) and inhibits glucose uptake, ultimately resulting in glucose metabolism disorders and increased susceptibility to type 2 diabetes¹⁴. Therefore, in-depth investigation and analysis of changes in amino acid metabolic profiles following exposure to PFOA and OBS can lay a foundation for elucidating their toxic mechanisms of action.

Based on the aforementioned research gaps and the pivotal biological implications of serum amino acid metabolism, we hypothesize that subchronic exposure to PFOA and OBS perturbs both mouse serum amino acid metabolic profiles significantly, and the two compounds exhibit distinct toxicological characteristics in disrupting specific amino acid species and metabolic pathways due to inherent differences in their chemical structures. In this study, an automatic amino acid analyzer was used to detect changes in the contents of free amino acids and their derivatives in mouse serum. Combined with statistical analysis, differential metabolites were screened to clarify the characteristics of the effects of subchronic exposure to PFOA and OBS on amino acid metabolism in mice, compare the metabolic differences in the toxic effects of these two PFCs, and provide a theoretical basis for research on the toxic mechanisms and health risk assessment of PFCs.

Materials and methods

Experimental animals and sample collection

Fifteen specific pathogen-free (SPF) male C57BL/6N mice, weighing 22 ± 2 g, were obtained from the National Experimental Animal Resource Centre (Shanghai, China). All mice were housed in an SPF animal facility with controlled temperature, stable relative humidity, and a 12 h light/12 h dark cycle. Standard laboratory chow and Milli-Q water were available ad libitum. After one week of acclimatization, the 15 mice were randomly divided into three groups (n = 5 per group): CON group, PFOA group (3 mg/(kg·d)), and OBS group (3 mg/(kg·d)). The exposure concentration of 3 mg/(kg·d) for both PFOA and OBS was selected based on the previous research results of our laboratory¹⁵. PFOA (CAS: 335-67-1) was purchased from Sigma Chemical Company (St. Louis, MO, USA), and OBS (CAS: 77061-68-8) was obtained from Shangflouro Company (China). The experiment lasted for 4 weeks, during which the body weight, food intake, and mental status of the mice were recorded daily. At the end of the experiment, blood samples were collected via retro-orbital puncture under isoflurane anesthesia. Briefly, mice were anesthetized with 5% isoflurane in an induction chamber and maintained with 2% isoflurane via a nose cone. Euthanasia was subsequently performed by cervical dislocation while the mice were under deep anesthesia.​ Blood samples were transferred into 1.5 mL centrifuge tubes and centrifuged at 3000 rpm for 10 min at 4 ℃. The separated serum was stored at -80 ℃ for subsequent analysis.

Detection of the contents of amino acids and their derivatives

A 1 mL serum sample was thoroughly mixed with 2.5 mL of 7.5% trichloroacetic acid solution, followed by centrifugation at 12,000 ×g and 4 °C for 15 min. The supernatant was collected, and its amino acid composition was analyzed using an automated amino acid analyzer (L-8900; Hitachi, Japan)¹⁶. A gradient elution program was adopted for the analysis with the following parameters: the injection volume of each sample was 20 μL, the total analysis cycle was 150 min, the column equilibration time was 35 min, and the detection wavelength was set at 570 nm (440 nm for proline). Quantification of individual free amino acids in the serum was performed using amino acid standards, all of which (including amino acid derivatives) were purchased from Merck Chemical Technology (Shanghai) Co., Ltd.

Data processing and statistical analysis

Student’s t-test, one-way analysis of variance (ANOVA), and tukey test were employed to analyze group differences using the SPSS 27.0 software (SPSS Inc., Chicago, IL, USA). A statistically significant difference was defined as a P-value < 0.05. All results were represented as mean ± SEM. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was performed using SIMCA-P 14.1 software (Umetrics AB, Sweden) to calculate the variable importance in projection (VIP) values. Data visualization was performed using the BioDeep website (https://www.biodeep.cn).

Results

Principal Component Analysis (PCA)

Amino acids exhibited significant dynamic variations following PFOA and OBS exposure. PCA revealed distinct intra-group clustering among the CON, PFOA, and OBS groups, which confirms consistent amino acid content and composition within each group. Notably, the CON group was well separated from the PFOA and OBS groups, indicating significant differences in amino acid profiles between the CON and exposure groups. In contrast, the sample distributions of the PFOA and OBS groups showed partial overlap, suggesting a high degree of similarity in their amino acid profiles (Fig. 1A). Variables in the loading plot reflect their contribution rates to sample discrimination under different treatments as well as the correlations among variables; the farther a variable is from the origin, the greater its contribution to group classification. We found that asparagine, 3-methylhistidine, serine, α-aminooctanedioic acid, leucine, and other amino acids were located relatively far from the origin, indicating that these amino acids had a higher contribution rate to the differentiation of samples treated with PFOA and OBS (Fig. 1B).

Fig. 1.

PCA score plot of mouse serum sample profiles. (A) PCA score plot showing the distribution of three groups. Ellipsoids represent 95% confidence intervals for each group. PC1 and PC2 explain 30.8% and 24.4% of the total variance, respectively; (B) PCA loading plot corresponding to (A), with labels indicating the contribution of individual amino acids to the separation of groups.

Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA)

OPLS-DA is a regression model established based on partial least squares, which enables efficient extraction of differential information between sample groups. All samples were distributed within the 95% confidence interval, indicating that the experiment had good stability and repeatability. A significantly high degree of separation was observed among the CON, PFOA, and OBS groups (Fig. 2A-D), suggesting that significant alterations occurred in amino acid metabolism following PFOA and OBS exposure. The model evaluation parameters (R² and Q²) obtained from cross-validation were both lower than the original R² and Q² values (Fig. 2E-G). These results demonstrated that the established OPLS-DA model was free from overfitting and could accurately and reliably characterize sample information to support subsequent analyses. Subsequently, biomarker screening was conducted using the criterion of variable importance in projection (VIP) > 1.0. A total of 23 differential amino acids and their derivatives were identified, including homocitrulline, homocysteine, sarcosine, threonine, β-alanine, 1-methylhistidine, ethanolamine, glutamine, hydroxyproline, α-aminoadipic acid, serine, phosphoethanolamine, asparagine, citrulline, α-aminosuberic acid, α-aminobutyric acid, phosphoserine, phenylalanine, 3-methylhistidine, leucine, isoleucine, valine, and tryptophan. Among these compounds, homocitrulline showed the highest VIP value of 1.372 (Fig. 3).

Fig. 2.

(A-D) Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) score plots of mouse sample profiles. The X-axis represents the degree of explanation of the first principal component, and the Y-axis represents the degree of explanation of the second principal component. The points represent the samples, and the colors represent the different groups.(E-G) Verification of OPLS-DA model. The R² and Q² points in the upper right corner represent the OPLS-DA model parameters of the true grouping of the samples.(A) CON vs PFOA vs OBS; (B) CON vs PFOA; (C) CON vs OBS; (D)PFOA vs OBS; (E) CON vs PFOA; (F) CON vs OBS; (G) PFOA vs OBS.

Fig. 3.

Variable Importance in Projection (VIP) plot of differential metabolites. The Y-axis represents the screened differential metabolites, while the X-axis denotes the corresponding VIP scores calculated from the orthogonal partial least squares discriminant analysis (OPLS-DA) model. Metabolites with VIP > 1 (the screening criterion for differential metabolites in this study, considered as key differential contributors) are marked in red, and those with VIP < 1 are marked in green.

Comparison of amino acid content in mouse serum under different treatments

Exposure to PFOA and OBS significantly altered the concentrations of several free amino acids in mouse serum (Table 1), whereas no statistically significant difference was observed in the total amino acid levels (P = 0.112).

Table 1.

Amino acid content in mouse serum under different treatments (µmol/L).

Items	Comparison of amino acid content			P-value
	CON	PFOA	OBS
Essential amino acid (EAA)
Lysine (Lys)	324.22±56.73	269.69±121.02	232.01±15.27	0.210
Tryptophan (Trp)	92.85±10.47	90.51±22.18	115.87±15.96	0.066
Phenylalanine (Phe)	56.40±5.50	69.99±12.38	61.93±4.72	0.068
Methionine (Met)	77.56±14.26	60.62±34.62	69.65±11.64	0.515
Threonine (Thr)	135.76±15.48ab	113.78±35.02b	170.25±8.12a	0.006
Isoleucine (Ile)	87.13±15.45	115.95±23.18	104.37±8.00	0.054
Leucine (Leu)	125.84±21.26b	168.32±35.67a	165.99±11.16a	0.032
Valine (Val)	186.97±26.70	233.67±42.93	224.89±10.49	0.065
Nonessential amino acid (NEAA)
Alanine (Ala)	270.43±62.17	225.23±60.94	276.39±31.81	0.292
Glutamic acid (Glu)	54.10±14.34	45.98±6.27	45.41±13.60	0.462
Serine (Ser)	90.72±21.00ab	82.41±19.22b	114.94±11.48a	0.034
Tyrosine (Tyr)	80.95±9.00	74.35±22.39	85.54±17.95	0.604
Asparagine (Asn)	30.82±6.98b	32.41±9.42b	44.90±3.34a	0.016
Cysteine (Cys)	225.08±42.93	195.55±52.61	200.79±23.05	0.505
Glutamine (Gln)	482.35±114.07b	567.34±55.38b	719.29±55.62a	0.002
Aspartic acid (Asp)	9.20±4.84	4.57±0.68	6.29±2.59	0.109
Glycine (Gly)	245.66±57.19	222.18±38.73	254.22±48.05	0.573
Proline (Pro)	108.25±15.27	89.38±35.63	98.57±6.84	0.447
Arginine (Arg)	116.81±23.28	85.55±30.56	91.95±27.01	0.199
Histidine (His)	49.94±6.68	58.23±8.89	53.57±3.00	0.184
Total amino acids	2851.04±543.6	2805.71±667.72	3136.82±329.7	0.112

In the same row, data with different small letter superscripts mean significant difference (P < 0.05), while with the same or no letter superscripts mean no significant difference (P > 0.05).

Among the essential amino acids, compared with the CON group, the levels of threonine and leucine in the PFOA and OBS expose groups exhibited significant differences (P < 0.05). Notably, several essential amino acids showed trends close to statistical significance: isoleucine levels exhibited a near-significant upward trend (P = 0.054); tryptophan (P = 0.066), phenylalanine (P = 0.068), and valine (P= 0.065) also showed trends toward alteration across the three groups, suggesting potential disturbances in branched-chain amino acid and aromatic amino acid metabolism that warrant further investigation. Furthermore, no statistically significant differences were detected in the levels of five other essential amino acids, including lysine, tryptophan, phenylalanine, methionine, and valine, across the three groups (P > 0.05).

For non-essential amino acids, the levels of serine, asparagine, and glutamine displayed significant differences among the three groups (P < 0.05). In contrast, no statistically significant variations were found in the levels of the remaining seven non-essential amino acids, such as alanine, glutamic acid, tyrosine, and aspartic acid, across the groups (P > 0.05).

Comparison of amino acid derivative content in mouse serum under different treatments

Significant differential changes were observed in the serum levels of free amino acid derivatives in the PFOA and OBS mice (Table 2). Specifically, the contents of citrulline, α-aminoadipic acid, α-aminosuberic acid, homocitrulline, total homocysteine, 1-methylhistidine, ethanolamine, phosphoserine, sarcosine, hydroxyproline, and β-alanine showed extremely significant differences among the three groups (P < 0.01). Concentrations of α-aminobutyric acid, 3-methylhistidine, and phosphoethanolamine differed significantly across groups (P < 0.05). In contrast, no statistically significant differences were noted in γ-aminobutyric acid, taurine, ornithine, cystathionine, carnosine, hydroxylysine, kynurenine, or anserine levels (P > 0.05).

Table 2.

Contents of amino acid derivatives in mouse serum under different treatments (µmol/L).

Items	Comparison of amino acid derivatives content			P-value
	CON	PFOA	OBS
Gamma-Aminobutyric Acid (GABA)	0.17±0.07	0.13±0.07	0.12±0.06	0.466
Taurine (Tau)	454.03±122.58	461.85±43.11	381.17±50.33	0.256
Citrulline (Cit)	54.50±7.15b	71.08±11.14ab	81.99±13.47a	0.006
Ornithine (Orn)	58.48±11.68	71.07±23.07	55.48±9.83	0.298
α-Aminoadipic Acid (AAA)	10.99±2.58a	6.20±0.80b	4.86±1.55b	<0.001
α-Aminobutyric Acid (AABA)	4.38±0.81ab	3.92±0.91b	5.78±1.16a	0.027
α-Aminosuberic Acid (Asu)	0.13±0.11b	0.47±0.17a	0.34±0.12a	0.006
Cystathionine (Cth)	1.27±0.21	1.11±0.29	1.32±0.23	0.370
Homocitrulline (Hcit)	0.52±0.07b	0.61±0.08b	1.26±0.10a	<0.001
Total Homocysteine (tHcy)	4.14±0.68b	8.44±1.45a	4.09±1.39b	<0.001
1-Methylhistidine (1-MHis)	4.40±0.86b	7.13±1.26a	5.39±0.64b	0.002
3-Methylhistidine (3-MHis)	6.49±1.10b	8.80±1.68ab	9.51±1.77a	0.024
Carnitine (Car)	1.00±0.29	0.86±0.09	0.95±0.16	0.516
Ethanolamine (Etn)	15.75±2.08a	13.25±1.32ab	10.69±1.95b	0.003
Phosphoserine (P-Ser)	0.13±0.04a	0.03±0.03b	0.10±0.05a	0.005
Hydroxylysine (Hylys)	0.34±0.09	0.25±0.14	0.19±0.05	0.093
Sarcosine (Sar)	1.14±0.16b	0.82±0.23b	1.66±0.31a	<0.001
Phosphoethanolamine (PEtn)	4.24±2.26a	1.20±0.35b	3.83±1.03a	0.012
Hydroxyproline (Hyp)	17.40±3.20a	10.19±2.77b	10.55±1.09b	0.001
β-Alanine (β-Ala)	9.39±2.43a	4.97±0.43b	4.21±0.88b	<0.001
Kynurenine (Kyn)	0.57±0.14	0.48±0.09	0.58±0.12	0.356
Anserine (Ans)	1.22±0.23	1.1±0.24	1.18±0.22	0.696

In the same row, data with different small letter superscripts mean significant difference (P < 0.05), while with the same or no letter superscripts mean no significant difference (P > 0.05).

Discussion

Amino acids serve as both raw materials for protein synthesis in the body and key metabolic intermediates. Alterations in their levels can directly reflect the body’s metabolic homeostasis¹⁷, nutritional status, and organ function^18,19. In this study, exposure to PFOA and OBS exerted no significant effect on the total amino acid content in mice, indicating that these two pollutants did not disrupt the overall balance of amino acid levels in the animals. However, they selectively interfered with the metabolic regulatory processes of specific amino acids and their derivatives, which may be attributed to the targeted regulatory effects of pollutants on specific metabolic pathways^6,20.

Essential amino acids are nutritionally indispensable substrates that cannot be endogenously synthesized by the body. As core raw materials for the synthesis of structural and functional proteins, they directly participate in physiological processes such as cell growth, tissue repair, and immune regulation. Additionally, essential amino acids are involved in energy metabolism and the synthesis of bioactive substances; their metabolic disorders can directly affect protein metabolism and the overall nutritional metabolic status of the body. Leucine, an essential branched-chain amino acid (BCAA), acts as a core regulator of protein synthesis and degradation, governing skeletal muscle protein metabolism²¹. It also participates in energy metabolism and the regulation of lipid-glucose homeostasis²², and plays crucial roles in physiological processes including growth and development, antioxidation, and anti-inflammation²³. Previous studies have demonstrated that PFOA exposure increases serum BCAA levels, disrupts their metabolic pathways, and may elevate the risk of type 2 diabetes mellitus²⁴. However, the effects of subchronic OBS exposure on the body remain unexplored. The results of this experiment showed that subchronic exposure to both PFOA and OBS significantly increased serum leucine levels in mice, which is consistent with the conclusions of the aforementioned studies. This suggests that both PFOA and OBS may increase the risk of type 2 diabetes mellitus. Furthermore, threonine, another essential amino acid, is a core component for maintaining the structure and function of the intestinal mucosa. It participates in protein synthesis, lipid metabolism, and immune regulation, and is critical for sustaining physiological homeostasis^24,25. This study revealed that serum threonine levels were significantly decreased in PFOA-exposed mice but significantly increased in OBS-exposed mice. This divergence suggests that although both compounds may disrupt intestinal or immune homeostasis, their underlying mechanisms are likely distinct. PFOA may lead to excessive threonine consumption through intestinal mucosal repair processes, whereas OBS may be associated with metabolic reprogramming related to immune activation^26–28. Isoleucine, another BCAA, exhibited a near-significant difference among the three groups, with slightly higher levels in the PFOA and OBS groups compared to the CON group. This, combined with the significant elevation of leucine levels, implies that different types of PFCs may interfere with BCAA metabolism to varying degrees.

Serine, a core substrate for one-carbon metabolism, sustains cell proliferation and redox homeostasis by mediating de novo nucleic acid synthesis and regulating epigenetic modifications. Dysregulation of serine metabolism can disrupt one-carbon flux, thereby driving metabolic reprogramming of tumor cells and promoting tumor progression and chemoresistance²⁹. Asparagine, acting as an amino acid exchange factor, provides critical metabolic support for tumor cell proliferation by regulating intracellular amino acid homeostasis and nutrient-sensing pathways. Abnormalities in its expression or function significantly affect the growth rate and malignant phenotype of tumor cells³⁰.In this study, the levels of serine and asparagine in the PFOA and OBS groups were significantly different from those in the CON group. Notably, the OBS group exhibited significantly higher levels of these two amino acids than both the CON and PFOA groups. These results indicate that exposure to PFOA and OBS can synergistically perturb the serine-asparagine metabolic network, potentially activating metabolic pathways closely associated with tumor initiation and progression, and thereby may enhance the risk of cellular malignant transformation. Among the two compounds, OBS exerted a more prominent upregulatory effect on serine and asparagine levels, suggesting that compared with PFOA, OBS may have a stronger capacity to regulate related metabolic pathways and induce abnormal cellular phenotypes. It is worth noting that the asparagine level in the PFOA group showed no significant difference from that in the CON group, indicating that PFOA had a weaker perturbing effect on asparagine metabolism than OBS. This finding suggests that the two compounds differ in their effects on specific amino acid metabolic pathways in mouse serum. Glutamine, the most abundant free amino acid in the body, is involved in energy supply, regulation of oxidative stress, maintenance of gut microbiota homeostasis, and promotion of intestinal epithelial cell proliferation. Additionally, glutamine regulates tight junction proteins and inhibits pro-inflammatory signaling pathways^31,32. This study found that glutamine levels were significantly increased following exposure to PFOA and OBS; however, no significant difference was observed between the PFOA and CON groups. These results imply that OBS exposure exerts a stronger interfering effect on glutamine metabolism in mouse serum than PFOA, leading to more severe physiological damage. Collectively, the above findings suggest that asparagine and glutamine may serve as stress-related metabolic biomarkers for OBS exposure.

As an important class of bioactive molecules, amino acid derivatives play a crucial role in physiological regulation and disease treatment. On the one hand, they serve as precursors for various important endogenous substances and directly participate in the regulation of life activities³³. For instance, studies have demonstrated that kynurenine, a key product of tryptophan metabolism, bidirectionally regulates the aging process and healthy lifespan of organisms by activating the aryl hydrocarbon receptor (AhR) signaling pathway. The activity of this pathway increases with age; excessive activation exacerbates oxidative stress, inflammation, and tissue degeneration, while moderate regulation or targeted inhibition of this pathway can maintain body homeostasis, delay aging, and provide novel targets for the prevention and treatment of age-related diseases³⁴. Taurine, a non-protein amino acid, possesses a unique sulfonic acid moiety that enables it to modulate a diverse array of cellular functions, including osmoregulation, antioxidation, ion homeostasis maintenance, and bile acid conjugation. Beyond these fundamental roles, taurine exerts potent anti-inflammatory effects, ameliorates diabetic phenotypes, and confers cardiovascular benefits potentially via the inhibition of the renin-angiotensin system³⁵. In this study, significant changes were observed in the levels of 14 amino acid derivatives in mouse serum following PFOA and OBS exposure. Previous studies have shown that PFOA exposure directly impairs the urea cycle, leading to metabolic dysfunction and hepatotoxicity³⁶.

Citrulline, as an intermediate product of the urea cycle³⁷, was presumed to accumulate in response to PFOA exposure, which is consistent with the findings of this study. α-Aminoadipic acid, an intermediate metabolite of lysine, regulates insulin secretion and serves as a predictor of diabetes³⁸. Additionally, α-aminoadipic acid enhances adipocyte thermogenesis by upregulating peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and uncoupling protein 1 (UCP1) mediated by β3-adrenergic receptor (β3AR) activation, thereby inducing higher energy expenditure. It also stimulates lipolysis by activating the β3AR signaling pathway to enhance the expression of hormone-sensitive lipase (HSL), suggesting that increasing endogenous α-aminoadipic acid levels may prevent obesity and diabetes^39,40. The results of this study revealed a significant decrease in α-aminoadipic acid content following PFOA and OBS exposure, suggesting that subchronic exposure to these two PFCs may be associated with an increased risk of obesity and diabetes in animals. Homocysteine is an intermediate product of methionine metabolism, and elevated tHcy levels are recognized as important biomarkers of oxidative stress, cardiovascular diseases⁴¹, and nervous system damage⁴². In this study, the tHcy content in the PFOA group was significantly higher than that in the CON group and OBS group, while no significant difference was observed between the OBS group and the CON group. This indicates that PFOA exposure is more likely to induce homocysteine accumulation in mice, thereby triggering the development of related diseases⁴³.

Subchronic exposure to PFOA and OBS also increased the levels of 1-methylhistidine and 3-methylhistidine in mouse serum; however, no significant difference in 1-methylhistidine content was found between the OBS group and the CON group. This suggests that PFOA may pose a higher risk of muscle tissue damage than OBS. Ethanolamine and phosphoethanolamine are important precursors of phospholipid metabolism, and changes in their levels directly reflect the status of phospholipid anabolism⁴⁴. In this study, ethanolamine content in both the PFOA group and OBS group was significantly lower than that in the CON group, whereas phosphoethanolamine content was significantly reduced only in the PFOA group compared to the CON group. These results indicate that both PFCs may inhibit the initial step of phospholipid synthesis, with PFOA exerting a stronger inhibitory effect. Phospholipids are key components of biological membranes; abnormalities in their anabolism directly affect the structure and function of cell membranes, disrupting cellular processes such as signal transduction and material transport, and ultimately contributing to the development of diseases⁴⁵. This may represent one of the core mechanisms underlying the toxic effects of PFCs. Furthermore, phosphoserine, an intermediate product of phosphatidylserine synthesis, was significantly decreased in the PFOA group but not in the OBS group, further confirming the specific interfering effect of PFOA on the phospholipid metabolic pathway.

Sarcosine is primarily involved in glycine metabolism and one-carbon unit metabolism, and is closely associated with energy metabolism and methylation reactions⁴⁶. In this study, sarcosine content in the OBS group was significantly higher than that in the CON group, while it was significantly lower in the PFOA group compared to the CON group. This may suggest that OBS could promote the conversion of glycine to sarcosine, whereas PFOA might inhibit this process. The two compounds thus exert opposite interference effects on the glycine metabolic pathway, which may be attributed to differences in their binding affinity or target sites with metabolic enzymes⁴⁷. Studies have shown that hydroxyproline is a characteristic amino acid of collagen, formed by the post-translational hydroxylation of proline in proteins (primarily collagen)⁴⁸. The reduced hydroxyproline content observed in this study may result from the inhibition of certain enzyme activities during the translation process by the two PFCs. β-Alanine is critical for preventing skin aging and intestinal damage, enhancing the compressive capacity of muscle cells, and suppressing age-related declines in memory and learning ability. A decrease in β-alanine content can induce or exacerbate various diseases by affecting the synthesis and physiological functions of key substances⁴⁹. Additionally, α-aminobutyric acid, a non-protein amino acid, has been shown to activate the AMP-activated protein kinase (AMPK)/silent information regulator 1 (SIRT1) signaling pathway in the liver, promoting fatty acid oxidation, inhibiting fat synthesis and inflammatory responses. It also regulates the composition of the intestinal flora, improves intestinal barrier function, and reduces endotoxin entry into the liver, thereby alleviating liver inflammation and damage⁵⁰. The results of this study indicated significant differences in serum α-aminobutyric acid content between the PFOA/OBS exposure groups and the CON group, suggesting that these two PFCs may contribute to the development of certain liver diseases. Notably, compared with the CON group, serum α-aminobutyric acid levels were significantly increased in the OBS group but significantly decreased in the PFOA group. This opposite pattern suggests that PFOA and OBS may exert distinct effects on the metabolic pathways involving this amino acid derivative, potentially through different mechanisms related to liver health. No significant differences were observed among the three groups in the levels of eight amino acid derivatives, including γ-aminobutyric acid, taurine, and ornithine. This indicates that the metabolic pathways of these amino acid derivatives are insensitive to PFOA and OBS exposure, which may be related to the substrate specificity of relevant metabolic enzymes or the body’s metabolic compensatory capacity⁵¹. The aforementioned differential results suggest that although both PFOA and OBS can interfere with the metabolism of amino acid derivatives, differences may exist in their target action pathways due to variations in their chemical structures. A limitation of this study is that only male mice were used. As sex differences can significantly influence amino acid metabolism and the toxicological responses to environmental pollutants, future studies should include both sexes to provide a more comprehensive understanding of the metabolic toxicity of PFOA and OBS⁵².

Conclusion

In summary, both PFOA and OBS exposure can significantly disrupt the serum amino acid metabolic profile of mice; however, differences exist in the metabolic pathways affected and the intensity of such interference. These specific metabolic alterations indicate that PFOA and OBS may exert toxic effects through distinct molecular mechanisms, with their potential health risks also differing in terms of focus. The findings of this study provide key clues for further elucidating the metabolic toxicity mechanisms of PFOA and OBS, and also offer a scientific basis for assessing their health risks and identifying specific biomarkers. Future studies should focus on validating these candidate biomarkers in larger cohorts, exploring the underlying molecular mechanisms, and investigating whether similar metabolic alterations occur in human populations exposed to these compounds.

Author contributions

**Xuezhen Guo** : Conceptualization, Methodology, Investigation, Visualization, Writing–Original draft. **Nana Jing** : Methodology, Investigation, Validation. **Shuang Liang** : Investigation, Validation. **Qingjie Wei** : Formal analysis, Validation. **Wenhui Cui** : Supervision. **Yingping Xiao** : Data curation, Fund acquisition, Investigation, Resources, Validation. **Hua Yang** : Conceptualizations, Project administration. **Fei Wang** : Conceptualization, Fund acquisition, Writing–Review & editing, Supervision.

Funding

This study was financially supported by the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products (Grant numbers: 2021DG700024-KF202402), National Natural Science Foundation of China (No. 32372907), the Leading Project of the “Three Agri-Priorities with Nine Directions” Science and Technology Collaboration Plans in Zhejiang Province (No. 2025SNJF101) and China Postdoctoral Science Foundation under Grant Number 2025M780248.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interest

The authors declare no competing interests.

Ethical

All animal procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang Academy of Agricultural Sciences. And all experiments were performed in accordance with the relevant guidelines and regulations.