Metabolomic changes following GenX and PFBS exposure in developing zebrafish

Abstract

Short chain per- and polyfluoroalkyl substances (PFAS), including hexafluoropropylene oxide dimer acid (GenX) and perfluorobutane sulfonate (PFBS), are replacement chemicals for environmentally persistent, long-chain PFAS. Although GenX and PFBS have been detected in surface and ground water worldwide, few studies provide information on the metabolic alterations or risks associated with their exposures. In this study, larval zebrafish were used to investigate the toxicity of early-life exposure to GenX or PFBS. Zebrafish were chronically exposed from 4 h post-fertilization (hpf) to 6 days post-fertilization (dpf) to 150 μM GenX or 95.0 μM PFBS. Ultra-high-performance liquid chromatography paired with high-resolution mass spectrometry was used to quantify uptake of GenX and PFBS into zebrafish larvae and perform targeted and untargeted metabolomics. Our results indicate that PFBS was 20.4 % more readily absorbed into the zebrafish larvae compared to GenX. Additionally, PFBS exposure significantly altered 13 targeted metabolites and 21 metabolic pathways, while GenX exposure significantly altered 1 targeted metabolite and 17 metabolic pathways. Exposure to GenX, and to an even greater extent PFBS, resulted in a number of altered metabolic pathways in the amino acid metabolism, with other significant alterations in the carbohydrate, lipid, cofactors and vitamins, nucleotide, and xenobiotics metabolisms. Our results indicate that GenX and PFBS impact the zebrafish metabolome, with implications of global metabolic dysregulation, particularly in metabolic pathways relating to growth and development.

1. Introduction

Per- and polyfluoroalkyl substances (PFAS) have emerged as contaminants of concern due to their bioaccumulation and potential adverse effects on animal and human health. PFAS are widely used in the manufacture of a range of consumer products, including non-stick or stain/water-resistant products (Sunderland et al., 2019). PFAS are a broad class of chemicals containing at least one fully fluorinated carbon atom and a functional head group (Rayne and Forest, 2009; Wallington et al., 2021). Due to the strength of the carbon fluorine bond, PFAS are recalcitrant to most chemical and biological degradation processes and persist in the environment (Glüge et al., 2020; Kucharzyk et al., 2017). Exposure to PFAS is associated with adverse effects to the immune, reproductive, and endocrine systems and diseases, including cancers in toxicology and epidemiological studies (Barbo et al., 2023; Podder et al., 2021; Sunderland et al., 2019).

Due to concerns about the potential health risks of PFAS exposure, long-chain PFAS (containing seven or more carbons in the perfluoroalkyl chain, including legacy perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS)) were voluntarily phased out of production in the early 2000s, resulting in increased production of replacement short-chain PFAS (containing fewer than seven carbons) (Janousek et al., 2019; Kabadi et al., 2020). Because of their shorter carbon chain length, these replacement chemicals were intended to be less persistent in the environment and are now used in industrial applications across the world (Bao et al., 2018; Wang et al., 2016). In 2022, the US environmental protection agency (EPA) set health advisory levels for four PFAS compounds in drinking water: PFOA and PFOS (0.004 ppt and 0.02 ppt, respectively) and their short chain replacement chemicals GenX (hexafluoropropylene oxide dimer acid (HFPO-DA)) and perfluorobutane sulfonate (PFBS) (10 and 2000 ppt, respectively) (USEPA, 2022). In March of 2023, the EPA proposed maximum contaminant level (MCL) goals for PFOA and PFOS in drinking water at 4 ppt each (USEPA, 2023). The MCL also proposed a Hazard Index, which sums the ratios of the water concentration to the level determined not to cause health effects for two short chain PFAS - GenX and PFBS – and two long chain PFAS - perfluorononanoic acid (PFNA) and perfluorohexane sulfonate (PFHxS) (USEPA, 2022, 2023). Concern over regulation of PFAS extends internationally; the European Union has strongly recommended the removal of most PFAS from industrial processes and has so far designated PFOS, PFHxS, and GenX as substances of very high concern (Brennan et al., 2021).

GenX and PFBS have been detected in surface and ground waters in many countries, with demonstrated bioaccumulation in nearby plants and animals (Blake et al., 2020; Taniyasu et al., 2003). The potential health effects of these replacement PFAS are largely unknown. However, preliminary animal studies suggest that there may be an association between GenX and PFBS exposure and alterations to thyroid function and reproduction, as well as low birth weight and stunted growth (Blake et al., 2020; Chen et al., 2018; Newsted et al., 2008). Zebrafish are widely used in toxicology research to understand the effects of xenobiotic exposure on the vertebrate exposome, which reflects the biological outcomes of exposures (Howe et al., 2013). Due to their rapid ex-utero embryonic development and genomic homology to humans, zebrafish are an excellent model organism for studying vertebrate embryonic development and health (Gaballah et al., 2020; Howe et al., 2013). Previous studies of early-life exposure of zebrafish to short-chain PFAS reveal alterations such as abnormal behavior, altered growth and development, cardiac edema, and developmental neurotoxicity (Gebreab et al., 2020; Min et al., 2023; Rericha et al., 2022). As short-chain PFAS become more prevalent in the environment, it is essential to study how biological systems may be affected by exposure to these chemicals.

Incorporating “omics”, such as genomics (the study of gene expression), transcriptomics (the study of RNA), proteomics (the study of proteins), and metabolomics (the study of metabolites and pathways), into zebrafish studies provides molecular insight into physiological alterations caused by chemical exposures (da Silva et al., 2021; Sukardi et al., 2010). Since metabolites are products of cellular processes, metabolic alterations directly indicate physiological and phenotypic alterations (da Silva et al., 2021; Lai et al., 2021). When applied to embryonic or larval zebrafish, metabolomics provides important insight into the effects of exposures on growth and development and molecular mechanisms for chemically induced toxicological endpoints (da Silva et al., 2021; Kossack et al., 2023).

Previous metabolomic studies of zebrafish exposure to the legacy long-chain PFAS, primarily PFOA and PFOS, have demonstrated alterations to the lipid, carbohydrate, and amino acid metabolomes (Cheng et al., 2016; Du et al., 2016; Gebreab et al., 2020). These studies included short-term and chronic exposure during several different life stages of zebrafish (embryos, larvae, and adults) and compared metabolomic effects of PFOA, PFOS, and next-generation PFAS (GenX, PFBS, PFO3TDA (perfluoro-3,6,9-trioxadecanoic acid)) (Cheng et al., 2016; Du et al., 2016; Gebreab et al., 2020; Min et al., 2023; Sant et al., 2018). One study exposed zebrafish embryos to either GenX or PFOA at concentrations up to 100 mg L⁻¹ and compared the embryonic toxicity and metabolic alterations induced by exposure to PFOA or GenX, finding that exposures to either chemical altered metabolites that are essential to liver function and development (Gebreab et al., 2020). A second study of embryonic exposure to 10 mg L⁻¹ PFBS indicated an induced stress response, neurotoxicity, and altered folate biosynthesis and methionine metabolisms (Hu et al., 2022). Both studies used very different exposure doses and examined different resulting metabolic impacts, thus knowledge of short-chain PFAS uptake and the use of similar exposure levels are needed to assess metabolomic alterations.

The objective of this study was to understand global metabolic alterations in developing zebrafish resulting from exposure to the short-chain PFAS GenX and PFBS. Using targeted PFAS analysis, GenX and PFBS uptake and body burden of exposed zebrafish larvae were quantified. Using untargeted metabolomics, global metabolic alterations induced by GenX and PFBS exposure were discovered, which provided insight into the molecular mechanisms of toxicity in early-life zebrafish exposure to short-chain PFAS. Based on the findings from untargeted analysis, targeted metabolomics was performed to quantify levels of 33 amino acid metabolites to compare between exposed and non-exposed larvae. The results of this study provide new information on short-chain PFAS uptake and metabolic alterations in exposed zebrafish larvae.

2. Materials and methods

2.1. Chemicals

All solutions were prepared using water purified by a Millipore Milli-Q^® Reference purification system (18.2 MΩ.cm at 25 °C and total organic content below 5 ppb). GenX (Ammonium perfluoro (2-methyl-3-oxahexanote), 95 % purity) used for the exposure solutions was purchased from Manchester Organics (Runcorn, England). Dimethyl sulfoxide (DMSO) and PFBS (perfluorobutane sulfonate) for exposure solutions were obtained from MilliporeSigma (St. Louis, MO). Isotopically labeled perfluorinated compound internal standards for quantitation of GenX and PFBS were purchased from Wellington Laboratories (Guelph, Ontario, Canada). All solvents, including ultra high-performance liquid chromatography (UHPLC) grade acetonitrile and water (99.9 %), were purchased from Fisher Scientific (Waltham, MA). Isotopically labeled metabolites (containing l-alanine, l-leucine, l-phenylalanine, l-tryptophan, and l-tyrosine) were obtained from Cambridge Isotopes (MSK-QC-KIT, Tewksbury, MA). Amino acid metabolite standards (A9906–10 mL) and other targeted metabolites (DL-3-Ure-idoisobutyric acid, N-Acetylputrescine hydrochloride, γ-Aminobutyric acid, l-Asparagine, l-Pipecolic acid, l-Glutamine, 3-Ureidopropionic acid) were purchased from Sigma Aldrich (St. Louis, MO).

2.2. Zebrafish spawning

All maintenance and experimental procedures involving zebrafish (Danio rerio) were approved by the Brown University institutional animal care and use committee (IACUC; 19–12–0003) and adhered to the National Institute of Health’s “Guide for the Care and Use of Laboratory Animals.” The zebrafish were kept in an aquatic habitat (Aquaneering Inc., San Diego, CA) with a 14:10 hour light-dark cycle (Westerfield, 2000) and water temperature control (28.5 ± 2 °C), filtration, purification, automatic pH and conductivity stabilization, and ultraviolet (UV) irradiation disinfection.

The night before spawning, adult AB (wildtype) zebrafish were acclimated to their new tank (1.7 L specialized, gridded spawning tanks (Techniplast, USA)) conditions to facilitate timed reproduction. A transparent partition was used to separate sexes. The tank partition was removed within 2 h of light cycle onset so that the zebrafish could spawn for 1 hour. Three independent spawning events were performed on separate days to acquire three biological replicates of each dosing experiment. Spawning occurred with separate parents on three different dates for each exposure compound (GenX, PFBS, or vehicle (egg water containing 0.1 % DMSO) to ensure an adequate number and varied parenthood of embryos. All embryos were collected in 100 mm non-treated culture petri dishes containing fresh egg water (60 mg L⁻¹ Instant Ocean Sea Salts; Aquarium Systems, Mentor, OF) after spawning. At 4 hpf, any embryos that did not show adequate cellular development by 4 manual screening for quality were discarded.

2.3. Exposure

Exposure Solutions:

Aqueous stock solutions of PFBS (64 mM) and GenX (41.48 mM) were prepared and validated at the start of the study. Two exposure solutions were prepared by diluting the stock solutions in fresh egg water in polypropylene tubes to obtain desired exposure concentrations of 150 μM (49.5 mg L⁻¹) for GenX and 100 μM (30.0 mg L⁻¹) for PFBS. Solutions were validated using targeted mass spectrometry (further described below) and the actual exposure solution concentrations were 150 μM (49.5 mg L⁻¹) for GenX and 95.0 μM (28.5 mg L⁻¹) for PFBS. For GenX, a previously published concentration between 100 and 200 μM demonstrated no physiological deformities or adverse effects in larval zebrafish at our desired timepoints (Gebreab et al., 2020); verification that no physiological deformities or excess mortality following exposure to 150 μM GenX by 6 days post-fertilization (dpf) was performed in order to proceed to collection and analysis. Likewise, the concentration selected for PFBS was also based on previously published work by Gaballah et al., who found that at 100 μM PFBS, exposed zebrafish had no physiological deformities or differences in locomotor activity in comparison with non-exposed groups (Gaballah et al., 2020). These exposure concentrations were verified not to produce physical deformities indicative of poor health, such as a bent spine or cardiac edema, by performing daily observations throughout the exposure period using a microscope.

Fish Exposure:

At 4 hpf, we plated 3 fertilized embryos per well in three sterile 24-well plates (ThermoFisher, Waltham, MA). Each well was dosed with 2 mL of either GenX, PFBS, or egg water with 0.1 % DMSO (vehicle control) (Fig. 1). DMSO was chosen as the vehicle because PFBS was dissolved in DMSO to generate a stock solution. GenX is unstable in DMSO and soluble in water (Gaballah et al., 2020); therefore, we dissolved GenX in purified deionized water. A previous study compared exposure solutions containing PFBS dissolved in DMSO and GenX in deionized water at the same concentrations and found that exposures resulted in changes of similar magnitudes (Gaballah et al., 2020). The plates were then incubated at 28.5 ± 1 °C incubator (Powers Scientific Inc., Pipersville, PA) from 4 hpf until 6 dpf. Fish were chronically exposed without replacement of the treatment solution, as PFBS and GenX are stable and highly persistent in aqueous solutions (Li et al., 2020a).

Fig. 1.

Experimental design for embryo spawning and exposure dosing for each exposure. Larvae were collected at 6df and then analyzed for PFAS body burden and metabolomics.

All embryos were manually dechorionated at 24 hpf to ensure un-protected exposure to toxicants throughout the protocol and placed back into the incubator until collection at 6 dpf (Henn and Braunbeck, 2011). Dechorionation was performed under a microscope to ensure no damage was done to the embryos during the process. For each exposure, 6 dpf larvae were pooled into 6 polypropylene centrifuge tubes (2 mL) of n = 10 larvae, which is a sufficient number of larvae for metabolomics based on the results of prior studies (da Silva et al., 2021; Kossack et al., 2023). Excess exposure solution was discarded from the larval pools, and then the pools were washed three times in laboratory-grade deionized water with all excess water removed between each round of washing. Samples were snap frozen in liquid nitrogen to be stored at −80 °C until ready for extraction. In total, 6 pools of 10 larvae were collected per biological replicate for each exposure group, resulting in 180 larvae per exposure group and 540 total larvae collected across all groups.

Sample Extraction:

30 μL of PFAS internal standard solution and 270 μL of acetonitrile containing an isotopically labeled metabolite internal standard solution were added to each sample. Samples were sonicated for 30 min until homogenized, and then all samples were centrifuged at 755 × g for 10 min using a Savant HSC-10K High-Speed Centrifuge. For metabolomics, 200 μL of the supernatant was transferred into an amber LC/MS vial containing a 300 μL glass insert. For PFAS analysis, the remaining 100 μL of supernatant was diluted in acetonitrile and spiked with internal standard to comprise 10 % of the total volume of the sample. These samples were transferred into 300 μL amber glass insert LC/MS vials. All samples were stored at 5 °C prior to analysis.

2.4. Analytical methods

PFAS Analysis:

Concentrations of GenX and PFBS were measured in the following solutions: (1) GenX and PFBS stock solutions, (2) diluted GenX and PFBS exposure solutions and matching vehicle (egg water containing 0.1 % DMSO), and (3) zebrafish larval extracts. To quantify PFBS or GenX in each solution, samples were diluted with UHPLC-grade acetonitrile containing 10 % isotope labeled internal standard.

GenX and PFBS concentrations were quantified using isotope dilution, based on a modified version of EPA Draft Method 1633 using a high-resolution Thermo QExactive HF-X Orbitrap MS equipped with a Vanquish ultra-high-performance liquid chromatograph (UHPLC—Orbitrap-HRMS) (USEPA, 2021). Analyte separation was achieved by injecting 20 μL of each sample onto a Thermo Hypersil Gold Vanquish C18 column (100 mm × 2.1 mm × 1.9 μm). Mobile phases consisted of 2 mM ammonium acetate in 5 % acetonitrile and 2 mM ammonium acetate in 100 % acetonitrile. The MS acquisitions were performed with negative electrospray ionization (ESI), in full scan data-dependent (MS-ddMS²) with an inclusion list for GenX, PFBS, and internal standards, and with 100–1500 m/z scan range. An HCD collision cell filled with N₂ gas (produced by a Peak Scientific Nitrogen Generator, Genius NM32LA) was used to perform MS² fragmentation. Additional details on the chromatography, source settings, and MS data collection parameters are provided in the SI (Table S1). PFAS concentrations were quantified using one quantification ion and eight-point calibration curves with concentrations ranging from 0 to 20,000 ng/L for each compound. Compound confirmation was performed using retention time and two confirming ions, when available. The Limits of Detection (LODs) were 42.1 ng/L for GenX and 34.6 ng/L for PFBS, which were determined from seven injections of calibration standards and multiplying the standard deviation of these injections by 3.143 (t_0.99 at 6 degrees freedom) divided by the calibration curve slope.

Targeted and Untargeted Metabolomics:

Both targeted and untargeted metabolomics were performed using the Thermo UHPLC–Orbitrap-HRMS. All samples were analyzed in triplicate using a randomized order in a single batch. All samples were analyzed using two chromatography methods, one with positive and negative ESI. For positive ESI (referred to as ESI+), a HILIC column (Thermo Syncronis HILIC 100 mm × 2.1 mm × 3 μm) was used. For negative ESI (referred to as ESI−), a C18 column (Thermo Hypersil Gold Vanquish, 100 mm × 2.1 mm × 1.9 μm) was used. Additional details on the chromatography, source settings, and dd-MS2 data collection parameters are provided in the SI (Table S2). For targeted metabolomics detected in the ESI+ method, a ten-point calibration curve ranging from 1.25 to 333 nM was used to quantify the metabolites in Table S3. Additionally, the limits of detection (LOD) for the targeted metabolites are in Table S3.

2.5. Untargeted metabolomics

Data Analysis:

XCalibur File Converter (ThermoFisher) was used to convert data files from .raw to .cdf format. R packages apLCMS and xMSanalyzer were used to produce m/z feature tables (Denison and Nagy, 2003; Uppal et al., 2013), which contained each feature (representative of a metabolite) in a row and the m/z value, paired retention time (seconds), and unitless feature areas in columns. All features containing greater than 20 % non-detection were removed. Each feature was then log2 transformed to normalize the data.

Statistical Analysis:

An Analysis of Covariance (ANCOVA) test was performed for each metabolomics data set (targeted and untargeted) for each exposure (GenX or PFBS) to test for between-group variance vs. within-group variance and account for the varying birth dates and parenthood of the zebrafish collected. For targeted data, ANCOVA results with an F-value > 0.05 were considered to be significantly altered metabolites (Mishra et al., 2019). In untargeted metabolomics, a false discovery rate (FDR) of 20 % was used to account for Type I errors in the comparisons (Kossack et al., 2023) and any feature above this threshold was used for pathway analysis.

Pathway Analysis:

Pathway analysis was performed using MetaboAnalyst’s Functional Analysis for zebrafish (Danio rerio (KEGG)) (Chong and Xia, 2018). Features were matched to metabolites with 5.0 ppm mass tolerance for ESI+ and ESI− data, including the feature F-value (equivalent to p-value in an ANCOVA) obtained from statistical analysis and retention time. The Mummichog algorithm was used with the default p-value cutoff of the top 10 % of features, which resulted in the following p-value (F-value) cut-offs: ESI+ GenX 0.25; ESI− GenX 0.1; ESI+ PFBS 0.05; ESI− PFBS 0.15 (Li et al., 2013). Our results include pathways that contained least 3 metabolites matched to features. Alterations to global metabolisms were mapped using the kyoto encyclopedia of genes and genomes (KEGG) (Kanehisa and Goto, 2000).

3. Results

3.1. Body burden

To establish levels of PFBS and GenX uptake, body burden per larva was determined for the six-day exposure period. GenX body burden in the exposure and vehicle groups was 26.6 ± 10.8 μg/embryo and 1.53 ± 0.495 μg/embryo, respectively (n = 10, Fig. 2). PFBS body burden in the exposure and vehicle groups was 19.3 ± 4.41 μg/embryo and 0.114 ± 0.0413 μg/embryo, respectively (n = 10, Fig. 2). Based on the exposure concentration in each exposure well (n = 3 embryos), the bioconcentration factors (BCFs), or the ratio of the concentration inside the zebrafish to the surrounding aqueous chemical concentration, were calculated to be 63.8 for PFBS and 50.6 for GenX (Table S4).

Fig. 2.

PFAS body burden of exposed to 95 μM PFBS or 150 μM GenX vs. vehicle groups at 6df. Each box plot represents 6 samples (each containing n = 10 larvae) that were analyzed by isotope dilution mass spectrometry in triplicate. Data were normalized to the number of larvae per sample, and the concentration is expressed per larvae.

3.2. Untargeted metabolomics: pathway analysis

A metabolome-wide association study (MWAS) was performed to examine alterations in the metabolome due to GenX or PFBS exposure in embryonic zebrafish. In GenX exposed embryos, 831 of 4554 detected features in the ESI− mode were significantly altered (FDR < 0.2), and 630 of 8517 detected features in ESI+ mode were significantly altered (FDR < 0.2) (Fig. 3). From the PFBS exposure, 596 of 4518 detected features in ESI− mode were significantly altered (FDR < 0.2), and 1897 of 8532 detected features in ESI+ mode were significantly altered (FDR < 0.2) (Fig. 3).

Fig. 3.

Volcano plots for PFBS (a,b) and GenX (c,d) exposed larva, with ionization mode indicated as ESI+/−. Each dot represents an individual feature (or metabolite). Log fold change was calculated for each log10-transformed metabolite detected in each exposure group by determining the difference between exposed and control levels of the metabolite and dividing the difference by the control value. The horizontal black line indicated on each graph represents a FDR= 0.2; all features (blue) above this line were significantly altered in the exposed groups in comparison to the vehicle group.

Features with −log10(p-value) above the 0.2 FDR threshold (Fig. 3) were used in pathway analysis to identify enriched pathways. For both GenX and PFBS exposures, enriched pathways were identified using three or more associated metabolites (features) for 42 metabolic pathways in total (Fig. 4). For the GenX exposure group, 32 total pathways were enriched with 18 unique to ESI+ mode, 13 overlapping between ESI+/− modes, and 1 unique to ESI− mode (Table S6). In the PFBS exposure group, 34 total pathways were enriched with 5 unique to ESI+ mode, 21 overlapping between ESI+/− modes, and 8 unique to ESI− mode (Table S6).

Fig. 4.

Enriched pathways from MetaboAnalyst pathway analysis, categorized by global metabolisms. The vertical black line represents −log10(0.05) (p-value = 0.05). Values to the right of the line on each plot represent significantly altered pathways (p-value < 0.05).

In the GenX exposed zebrafish, 17 out of 32 pathways were significantly (p > 0.05) enriched in at least one ionization mode. In the PFBS exposed zebrafish, 21 out of 34 pathways were significantly enriched in at least one of the two ionization modes. Enriched pathways were grouped into nine different global metabolomic categories (Table S6): amino acid metabolism, carbohydrate metabolism, energy metabolism, genetic information processing, glycan biosynthesis, lipid metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, and xenobiotics metabolism. Across all ionization modes and exposures, the global amino acid metabolism contained the greatest number of enriched (15) and significantly enriched (14 out of 15 in at least one exposure group and ionization mode) pathways.

3.3. Targeted metabolic pathway analysis

Based on the results from untargeted analysis, in which the largest number of significantly altered pathways occurred in the amino acid metabolism, targeted analysis was performed on 33 amino acid metabolites to further investigate metabolic changes in the larvae. In control and exposed larvae, 15 out of the 33 targeted metabolites were detected, including glutamic acid, isoleucine, l-alanine and B-alanine, l-citrulline, l-creatinine, l-tryptophan, methionine, phenylalanine, proline, sarcosine, serine, taurine, threonine, tyrosine, and valine (Fig. 5; Table S5). Concentrations ranged from <0.1 μg/larva to 11.1 μg/larva across all samples and targeted metabolites.

Fig. 5.

Targeted metabolites detected in GenX and PFBS exposed zebrafish larva at 6df (each exposure group contained n = 6 groups of 10 larvae). * indicates significant alteration from the vehicle for PFBS exposure. ^ indicates significant alteration from the vehicle for GenX exposure. To assess significance, a p-value less than 0.05 was used.

In GenX exposed fish, one targeted metabolite, l-citrulline, was significantly decreased (p < 0.05) (Fig. 5; Table S5). In PFBS exposed fish, 11 targeted metabolites (isoleucine, l-alanine and B-alanine, l-citrulline, l-tryptophan, methionine, phenylalanine, proline, sarcosine, serine, threonine, and tyrosine) were significantly decreased (p < 0.05), while two metabolites were significantly increased (L-creatinine and taurine) (p < 0.05).

4. Discussion

4.1. Body burden

Overall, the body burden data demonstrate high uptake of both GenX and PFBS within zebrafish embryos, as greater than 80 % of the applied mass of each compound within exposure solutions was absorbed by the larvae. However, the difference in body burden between each compound provide insight into how readily each compound entered the zebrafish tissue. Despite a 73.7 % higher GenX exposure dose than PFBS (mg L⁻¹ comparison), GenX bioconcentration factor (BCF) was lower than PFBS BCF. Higher BCF result indicate a more bioaccumulative substance (Wassenaar et al., 2020; Winston H. Hickox, 2000). In comparison to other PFAS BCFs in zebrafish, BCFs for both GenX and PFBS were similar, but lower, to PFOA (100) and PFHxS (110) and less than PFOS (2000) (Menger et al., 2020; Tal and Vogs, 2021). This was expected, considering GenX and PFBS are a PFOA alternative and a short chain sulfonate, respectively, and are much less bioaccumulative than PFOA and PFOS (Brandsma et al., 2019; Burkhard, 2021). The discrepancy between exposure dose uptake and body burden suggests that PFBS more readily entered the tissue of exposed zebrafish larvae than GenX or that GenX is more readily excreted from the larvae. Interestingly, comparison between body burdens and metabolic pathway alterations demonstrates that PFBS exposure resulted in a greater number of both significantly altered targeted metabolites and significantly altered pathways despite a lower exposure dose and body burden. The greater number of metabolic alterations observed in the PFBS group may indicate a higher toxicity of PFBS exposure as compared to GenX exposure.

4.2. Biological significance of impacted pathways

GenX Exposure:

GenX exposure resulted in 32 altered pathways, of which 17 were significantly altered (description of each of the significantly altered pathways can be found in Table S6). The significantly altered pathways in the GenX exposed zebrafish fell into all nine global metabolisms: amino acid metabolism, carbohydrate metabolism,energy metabolism, genetic information processing, lipid metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, and xenobiotics biodegradation and metabolism (Table S7). Disruptions to these pathways, individually or in combination with each other, could affect many organ systems and biological processes, such as response to oxidative stress or disease, liver function, and maintenance of metabolic processes through cellular development. In humans, alterations to several of the identified significantly altered pathways are associated with severe diseases, including neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases (purine metabolism), cancerous tumor growth (glycine, serine, and threonine metabolism), and metabolic syndrome (association with weight gain, Type 2 Diabetes, obesity (alanine, aspartate, and glutamate metabolism; cysteine and methionine metabolism; glycerolipid metabolism; glycine, serine, and threonine metabolism) (Amelio et al., 2014; Ansoleaga et al., 2015; Chen et al., 2022; Garcia-Esparcia et al., 2015; Li et al., 2020b). A previous study of zebrafish embryonic and larval exposure to PFOS and other non-PFAS endocrine disruptors found that alterations to alanine and proline metabolism indicated oxidative stress and imbalances in homeostasis and energy metabolisms (Ortiz-Villanueva et al., 2018). Another study found that PFBS exposure caused dysregulation of lipid homeostasis, which disrupted embryonic development, energy homeostasis, and pancreatic organogenesis (Sant et al., 2018). Further, alterations to the methionine metabolism had adverse effects on liver formation during zebrafish development (Liu et al., 2016). Our observed perturbations to metabolic pathways suggest metabolic dysregulation in the zebrafish embryos, with potential physiological implications ranging from liver development to tissue maintenance.

In addition to the functions listed in Table S6, several significantly altered pathways are associated with essential amino acids, or amino acids that must be consumed in animals’ diets for proper development and maintenance of cells, tissues, and in bodily functions. From the list of impacted metabolic pathways in Table S6, the following are nutritionally essential amino acids: cysteine, histidine, lysine, methionine, threonine, tryptophan, and tyrosine (Watts and D’Abramo, 2021). Disruption of essential amino acid pathways for processing and utilizing amino acids in the body can have effects on growth, survival, reproduction, and overall health of the animal, which indicates that metabolic pathways essential to development and health of the zebrafish have been impacted by exposure to GenX in this study (Watts and D’Abramo, 2021). In combination with the impacts of altered metabolisms that affect cellular development and growth, protein synthesis and muscular maintenance, and metabolic homeostasis, exposure to GenX during the embryonic and larval stages may have impacts on the proper development and growth of the zebrafish. These results are supported by transcriptomics data from a similar exposure study in zebrafish, which reported alterations to gene expression associated with energy homeostasis, nervous system development, and musculoskeletal function when larvae were exposed to GenX (Gong et al., 2023). The similarities in altered pathways across two “omics” approaches indicate that GenX may affect zebrafish across multiple biosynthetic systems.

PFBS Exposure:

Despite lower body burden in the PFBS exposed zebrafish, PFBS exposure resulted in a greater number of significantly altered pathways and targeted metabolites. Significant overlaps exist between significantly altered pathways from the GenX exposure group and PFBS exposure group; there were 12 significantly altered pathways uniquely in the PFBS exposure group in addition to the 9 shared with the GenX exposed zebrafish (Table S6). Significantly altered pathways for the PFBS exposed zebrafish, including overlap with the GenX exposed zebrafish, were in 8 global metabolisms; amino acid metabolism, carbohydrate metabolism, genetic information processing, lipid metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, and xenobiotics biodegradation and metabolism (Table S6). PFBS exposure resulted in 12 additional significantly altered targeted metabolites with the only overlap with the GenX exposure group being l-Citrulline, which was significantly downregulated in both exposures (Table S5).

Several metabolic pathways affected in the PFBS exposure group further contribute to metabolic syndrome, including alanine, aspartate, and glutamate metabolism, arginine biosynthesis, TCA Cycle, and valine, leucine, and isoleucine biosynthesis (Chen et al., 2022). Several metabolic pathway alterations overlap with significantly altered metabolites from targeted analysis, including alterations to levels of alanine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. There are also several additional functions impacted by the pathways that were altered uniquely in the PFBS exposure group, specifically alterations in energy/carbohydrate metabolism and its related amino acid pathways (TCA Cycle; glyoxylate and dicarboxylate metabolism; alanine, aspartate, and glutamate metabolism) and effects on the lipid metabolism. Previous zebrafish toxicology studies indicate that alterations to the lipid (fatty acid) metabolism affects lipid creation and utilization in the liver, and disturbances to the carbohydrate metabolism may indicate insulin resistance, associated with diabetes (Du et al., 2016; Sant et al., 2018; Teng et al., 2018). In humans, diabetes can be a result of metabolic syndrome, so alterations to the amino acid metabolisms affected in both exposures as well as the alterations to the lipid metabolism observed in the PFBS exposure group may indicate chronic perturbations to organ functions and bodily regulation (Chen et al., 2022). Results from a study of PFBS exposure in zebrafish embryos, which incorporated multi-omics analyses (proteomics, transcriptomics, and metabolomics), reported alterations to genetic and proteomic pathways, leading to lipid formation, oxidative stress response, and neurotoxicity during development (Min et al., 2023). Another study that investigated PFBS exposure in zebrafish found disruptions in regulation of lipid homeostasis using RNA-Seq data after 4 dpf (Sant et al., 2018). The fact that proteomics, transcriptomics, and metabolomics all demonstrate alterations to The fact that proteomics, transcriptomics, and metabolomics all demonstrate alterations to similar pathways indicates that PFBS may induce global dysregulation of macromolecule usage in zebrafish, with potential long-term impacts as indicated by links between pathway perturbations and disease.

It is also important to consider the implications of these results for human health, especially if contamination of water with replacement PFAS compounds such as PFBS and GenX becomes more widespread. The primary route of exposure to PFAS is through consumption of contaminated food and drinking water, and studies show that consumption of freshwater fish contaminated with PFAS can cause a direct increase in blood serum levels of PFAS in humans (Barbo et al., 2023). Considering the high uptake of GenX and PFBS by zebrafish in this study, ingestion of these compounds could pose dangers to human health through an increase in blood serum levels of GenX and PFBS. Not only would consumption of contaminated fish pose risks to the consumer, but the effects observed in this study would likely be most relevant to in utero exposure to PFAS. Fetal exposure to PFAS during pregnancy can cause the fetus to be at higher risk for metabolic syndrome, which is consistent with metabolites altered in this study, as well as thyroid dysfunction and kidney disease (Blake and Fenton, 2020). Using zebrafish as a toxicological model for both fish uptake of novel PFAS as well as potential human health effects demonstrates that adverse health effects are possible in vertebrate populations exposed to GenX and PFBS.

4.3. Study limitations

The single analyte exposure paradigm used in this study was different from environmentally relevant exposures to these chemicals. Across the US, freshwater fish are chronically exposed to combinations of PFAS compounds (concentrations depending on geographic location) at lower exposure doses than used in this study (Barbo et al., 2023). Even in the Cape Fear River, a site of known GenX contamination due to an upstream chemical plant, the maximum concentration of GenX in a water quality study was 4.56 μg L⁻¹, with an average of 630 ng L⁻¹, indicating that the 49.5 mg L⁻¹ exposure dose used in this study was an order of magnitude higher than GenX concentrations found in surface waters across the US (Sun et al., 2016). Although our exposure doses may not be representative of chronic exposure levels, the results are relevant to embryonic or early life exposure to GenX or PFBS. Therefore, developmental metabolome alterations from this study could be detected to a lesser extent at lower exposure doses observed in aquatic environments.

This study also included several limiting factors that did not allow for further comparison or analysis of the toxicity and metabolic effects of GenX and PFBS. Firstly, the exposure doses were based on mortality and/or physiological alterations that occurred due to exposure; however, the concentrations used in exposure were not equal, nor were there multiple concentrations to compare the level of toxicity of each compound to the embryos. For example, it would be useful to assess exposure doses for each compound leading to a similar magnitude or number of metabolic, as we observed a greater number of metabolic alterations in the PFBS exposure group with a different exposure dose. Additionally, this study focused on the individual effects of each compound; future studies would ideally assess exposure to a mixture of PFBS and GenX as well as other short-chain PFAS. While there are limitations to this study, including unequal exposure doses and lack of multiple dosing concentrations or additional dosing compounds, the study as a whole provides a basis for future studies that can provide additional insight into the toxicity of GenX and PFBS to zebrafish and other vertebrates.

5. Conclusions

Overall, this study provides an understanding of metabolic alterations induced by exposure to GenX or PFBS in zebrafish embryos. These exposures resulted in no physiological deformities or excess mortality of the zebrafish over a 6-day exposure period. The body burden assessment indicated that PFBS is more readily absorbed in zebrafish larvae compared to GenX. Targeted and untargeted metabolomics were used to understand alterations to the zebrafish metabolome induced by exposure to GenX and PFBS, and PFBS exposure induced a greater number of significantly altered metabolites and metabolic pathways than GenX exposure. Altered metabolic pathways in both GenX and PFBS exposed zebrafish indicate dysregulation of energy homeostasis, growth-related metabolites, and metabolites associated with lipid creation in the liver. These results indicate that developmental exposure to GenX or PFBS may disrupt cellular development and metabolic regulation in multiple organ systems. Further studies on combined effects of GenX, PFBS, and other short-chain replacement PFAS will provide greater detail on potential health effects of ingestion of these chemicals for both humans and other animals.

Data availability

Data will be made available on request.