Disclosing Environmental Ligands of L-FABP and PPARγ: Should We Re-evaluate the Chemical Safety of Hydrocarbon Surfactants?

Chemical contaminants can cause adverse effects by binding to the liver-fatty acid binding protein (L-FABP) and peroxisome proliferator-activated nuclear receptor γ (PPARγ), which are vital in lipid metabolism. However, the presence of numerous compounds in the environment has hindered the identification of their ligands, and thus only a small portion have been discovered to date. In this study, protein Affinity Purification with Nontargeted Analysis (APNA) was employed to identify the ligands of L-FABP and PPARγ in indoor dust and sewage sludge. A total of 83 nonredundant features were pulled-out by His-tagged L-FABP as putative ligands, among which 13 were assigned as fatty acids and hydrocarbon surfactants. In contrast, only six features were isolated when His-tagged PPARγ LBD was used as the protein bait. The binding of hydrocarbon surfactants to L-FABP and PPARγ was confirmed using both recombinant proteins and reporter cells. These hydrocarbon surfactants, along with >50 homologues and isomers, were detected in dust and sludge at high concentrations. Fatty acids and hydrocarbon surfactants explained the majority of L-FABP (57.7 ± 32.9%) and PPARγ (66.0 ± 27.1%) activities in the sludge. This study revealed hydrocarbon surfactants as the predominant synthetic ligands of L-FABP and PPARγ, highlighting the importance of re-evaluating their chemical safety.

Building the Environmental Chemical-Protein Interaction Network (eCPIN): An Exposome-Wide Strategy for Bioactive Chemical Contaminant Identification.

Although advancements in nontargeted analysis have made it possible to detect hundreds of chemical contaminants in a single run, the current environmental toxicology approaches lag behind, precluding the transition from analytical chemistry efforts to health risk assessment. We herein highlighted a recently developed "top-down" bioanalytical method, protein Affinity Purification with Nontargeted Analysis (APNA), to screen for bioactive chemical contaminants at the "exposome-wide" level. To achieve this, a tagged functional protein is employed as a "bait" to directly isolate bioactive chemical contaminants from environmental mixtures, which are further identified by nontargeted analysis. Advantages of this protein-guided approach, including the discovery of new bioactive ligands as well as new protein targets for known chemical contaminants, were highlighted by several case studies. Encouraged by these successful applications, we further proposed a framework, i.e., the environmental Chemical-Protein Interaction Network (eCPIN), to construct a complete map of the 7 billion binary interactions between all chemical contaminants (>350,000) and human proteins (∼20,000) via APNA. The eCPIN could be established in three stages through strategically prioritizing the ∼20,000 human proteins, such as focusing on the 48 nuclear receptors (e.g., thyroid hormone receptors) in the first stage. The eCPIN will provide an unprecedented throughput for screening bioactive chemical contaminants at the exposome-wide level and facilitate the identification of molecular initiating events at the proteome-wide level.

Proteome-wide effects of naphthalene-derived secondary organic aerosol in BEAS-2B cells are caused by short-lived unsaturated carbonyls.

Exposure to air pollution causes adverse health outcomes, but the toxicity mechanisms remain unclear. Here, we investigated the dynamic toxicities of naphthalene-derived secondary organic aerosol (NSOA) in a human bronchial epithelial cell line (BEAS-2B) and identified the chemical components responsible for toxicities. The chemical composition of NSOA was found to vary with six simulated atmospheric aging conditions (C1-C6), as characterized by high-resolution mass spectrometry and ion mobility mass spectrometry. Global proteome profiling reveals dynamic evolution in toxicity: Stronger proteome-wide impacts were detected in fresh NSOA, but the effects declined along with atmospheric aging. While Nrf2-regulated proteins (e.g., NQO1) were significantly up-regulated, the majority (78 to 97%) of proteins from inflammation and other pathways were down-regulated by NSOA exposure (e.g., Rho GTPases). This pattern is distinct from the reactive oxygen species (ROS)-mediated toxicity pathway, and an alternative cysteine reaction pathway was revealed by the decreased abundance of proteins (e.g., MT1X) prone to posttranslational thiol modification. This pathway was further validated by observing decreased Nrf2 response in reporter cells, after preincubating NSOA with cysteine. Ethynyl-naphthalene probe was employed to confirm the alkylation of cellular proteome thiols on the proteome-wide level by fresh NSOA via in-gel fluorescence imaging. Nontarget analysis identified several unsaturated carbonyls, including naphthoquinones and hydroxylated naphthoquinones, as the toxic components responsible for cysteine reactivity. Our study provides insights into the dynamic toxicities of NSOA during atmospheric aging and identifies short-lived unsaturated carbonyls as the predominant toxic components at the posttranslational level.

Significant Reductive Transformation of 6:2 Chlorinated Polyfluorooctane Ether Sulfonate to Form Hydrogen-Substituted Polyfluorooctane Ether Sulfonate and Their Toxicokinetics in Male Sprague-Dawley Rats.

6:2 chlorinated polyfluorooctane ether sulfonate (6:2 Cl-PFESA) was previously shown to undergo limited dechlorination in rainbow trout to yield 6:2 hydrogen-substituted polyfluorooctane ether sulfonate (6:2 H-PFESA) as the sole metabolite. However, the biotransformation susceptibility of 6:2 Cl-PFESA has not been investigated in mammals and the biological behavior of 6:2 H-PFESA has not been defined in any species. We investigated the respective transformation products of 6:2 Cl-PFESA and 6:2 H-PFESA and their toxicokinetic properties in male Sprague-Dawley rats as a mammalian model. 6:2 H-PFESA was the sole detectable metabolite of 6:2 Cl-PFESA, with a transformation percentage of 13.6% in rat liver, but it resisted further degradation. 6:2 Cl-PFESA also transformed to 6:2 H-PFESA in reductive rat liver S9 incubations but remained stable under oxidative conditions, suggesting a reductive enzyme-dependent transformation pathway. 6:2 Cl-PFESA was more enriched in lipid-rich tissues, while 6:2 H-PFESA was more prone to cumulative urinary excretion. From this perspective, it may suggest a detoxification mechanism for organisms to form the less hydrophobic 6:2 H-PFESA to alleviate total burdens. To date, 6:2 Cl-PFESA was the second perfluoroalkyl acid reported to undergo biotransformation in mammals. The toxicokinetic properties determined for 6:2 Cl-PFESA and 6:2 H-PFESA in blood and urine were found to be structure and dose dependent.

Anaerobic Microbial Dechlorination of 6:2 Chlorinated Polyfluorooctane Ether Sulfonate and the Underlying Mechanisms.

The microbial transformation potential of 6:2 chlorinated polyfluorooctane ether sulfonate (6:2 Cl-PFESA) was explored in anaerobic microbial systems. Microbial communities from anaerobic wastewater sludge, an anaerobic digester, and anaerobic dechlorinating cultures enriched from aquifer materials reductively dechlorinated 6:2 Cl-PFESA to 6:2 hydrogen-substituted polyfluorooctane ether sulfonate (6:2 H-PFESA), which was identified as the sole metabolite by non-target analysis. Rapid and complete reductive dechlorination of 6:2 Cl-PFESA was achieved by the anaerobic dechlorinating cultures. The microbial community of the anaerobic dechlorinating cultures was impacted by 6:2 Cl-PFESA exposure. Organohalide-respiring bacteria originally present in the anaerobic dechlorinating cultures, including Geobacter, Dehalobacter, and Dehalococcoides, decreased in relative abundance over time. As the relative abundance of organohalide-respiring bacteria decreased, the rates of 6:2 Cl-PFESA dechlorination decreased, suggesting that the most likely mechanism for reductive dechlorination of 6:2 Cl-PFESA was co-metabolism rather than organohalide respiration. Reductive defluorination of 6:2 Cl-PFESA was not observed. Furthermore, 6:2 H-PFESA exhibited 5.5 times lower sorption affinity to the suspended biosolids than 6:2 Cl-PFESA, with the prospect of increased mobility in the environment. These results show the susceptibility of 6:2 Cl-PFESA to microbially mediated reductive dechlorination and the likely persistence of the product, 6:2 H-PFESA, in anaerobic environments.

Defining the Chemical Additives Driving In Vitro Toxicities of Plastics.

Increases in the global use of plastics have caused concerns regarding potential adverse effects on human health. Plastic products contain hundreds of potentially toxic chemical additives, yet the exact chemicals which drive toxicity currently remain unknown. In this study, we employed nontargeted analysis and in vitro bioassays to identify the toxicity drivers in plastics. A total of 56 chemical additives were tentatively identified in five commonly used plastic polymer pellets (i.e., PP, LDPE, HDPE, PET, and PVC) by employing suspect screening and nontargeted analysis. Phthalates and organophosphates were found to be dominant in PVC pellets. Triphenyl phosphate and 2-ethylhexyl diphenyl phosphate accounted for a high amount (53.6%) of the inhibition effect of PVC pellet extract on human carboxylesterase 1 (hCES1) activity. Inspired by the high abundances of chemical additives in PVC pellets, six different end-user PVC-based products including three widely used PVC water pipes were further examined. Among them, extracts of PVC pipe exerted the strongest PPARγ activity and cell viability suppression. Organotins were identified as the primary drivers to these in vitro toxicities induced by the PVC pipe extracts. This study clearly delineates specific chemical additives responsible for hCES1 inhibition, PPARγ activity, and cell viability suppression associated with plastic.

Per- and Polyfluoroalkyl Substances in North American School Uniforms.

We analyzed 72 children's textile products marketed as stain-resistant from US and Canadian stores, particularly school uniforms, to assess if clothing represents a significant route of exposure to per- and polyfluoroalkyl substances (PFAS). Products were first screened for total fluorine (total F) using particle-induced γ-ray emission (PIGE) spectroscopy (n = 72), followed by targeted analysis of 49 neutral and ionic PFAS (n = 57). PFAS were detected in all products from both markets, with the most abundant compound being 6:2 fluorotelomer alcohol (6:2 FTOH). Total targeted PFAS concentrations for all products collected from both countries ranged from 0.250 to 153 000 ng/g with a median of 117 ng/g (0.0281-38 100 µg/m2, median: 24.0 µg/m2). Total targeted PFAS levels in school uniforms were significantly higher than in other items such as bibs, hats, stroller covers, and swimsuits, but comparable to outdoor wear. Higher total targeted PFAS concentrations were found in school uniforms made of 100% cotton than synthetic blends. Perfluoroalkyl acids (PFAAs) precursors were abundant in school uniforms based on the results of hydrolysis and total oxidizable precursor assay. The estimated median potential children's exposure to PFAS via dermal exposure through school uniforms was 1.03 ng/kg bw/day. Substance flow analysis estimated that ∼3 tonnes/year (ranging from 0.05 to 33 tonnes/year) of PFAS are used in US children's uniforms, mostly of polymeric PFAS but with ∼0.1 tonne/year of mobile, nonpolymeric PFAS.

Quantitative Chemical Proteomics Reveals Interspecies Variations on Binding Schemes of L-FABP with Perfluorooctanesulfonate.

Evaluating interspecies toxicity variation is a long-standing challenge for chemical hazard assessment. This study developed a quantitative interspecies thermal shift assay (QITSA) for in situ, quantitative, and modest-throughput investigation of chemical-protein interactions in cell and tissue samples across species. By using liver fatty acid binding protein (L-FABP) as a case study, the QITSA method was benchmarked with six per- and polyfluoroalkyl substances, and thermal shifts (ΔTm) were inversely related to their dissociation constants (R2 = 0.98). The QITSA can also distinguish binding modes of chemicals exemplified by palmitic acid. The QITSA was applied to determine the interactions between perfluorooctanesulfonate (PFOS) and L-FABP in liver cells or tissues from humans, mice, rats, and zebrafish. The largest thermal stability enhancement by PFOS was observed for human L-FABP followed by the mouse, rat, and zebrafish. While endogenous ligands were revealed to partially contribute to the large interspecies variation, recombinant proteins were employed to confirm the high binding affinity of PFOS to human L-FABP, compared to the rat and mouse. This study implemented an experimental strategy to characterize chemical-protein interactions across species, and future application of QITSA to other chemical contaminants is of great interest.

Toxicokinetics of Brominated Azo Dyes in the Early Life Stages of Zebrafish (Danio rerio) Is Prone to Aromatic Substituent Changes.

Brominated azo dyes (BADs) have been identified as predominant indoor brominated pollutants in daycare dust; thus, their potential health risk to children is of concern. However, the toxicities of BADs remain elusive. In this study, the toxicokinetics of two predominant BADs, Disperse Blue 373 (DB373) and Disperse Violet 93 (DV93), and their suspect metabolite 2-bromo-4,6-dinitroaniline (BDNA) was investigated in embryos of zebrafish (Danio rerio). The bioconcentration factor of DV93 at 120 hpf is 6.2-fold lower than that of DB373. The nontarget analysis revealed distinct metabolism routes between DB373 and DV93 by reducing nitro groups to nitroso (DB373) or amine (DV93), despite their similar structures. NAD(P)H quinone oxidoreductase 1 (NQO1) and pyruvate dehydrogenase were predicted as the enzymes responsible for the reduction of DB373 and DV93 by correlating time courses of the metabolites and enzyme development. Further in vitro recombinant enzyme and in vivo inhibition results validated NQO1 as the enzyme specifically reducing DB373, but not DV93. Global proteome profiling revealed that the expression levels of proteins from the "apoptosis-induced DNA fragmentation" pathway were significantly upregulated by all three BADs, supporting the bioactivation of BADs to mutagenic aromatic amines. This study discovered the bioactivation of BADs via distinct eukaryotic enzymes, implying their potential health risks.

Nontarget Screening of Per- and Polyfluoroalkyl Substances Binding to Human Liver Fatty Acid Binding Protein.

More than 1000 per- and polyfluoroalkyl substances (PFASs) have been discovered by nontarget analysis (NTA), but their prioritization for health concerns is challenging. We developed a method by incorporating size-exclusion column co-elution (SECC) and NTA, to screen PFASs binding to human liver fatty acid binding protein (hL-FABP). Of 74 PFASs assessed, 20 were identified as hL-FABP ligands in which eight of them have high binding affinities. Increased PFAS binding affinities correlate with stronger responses in electrospray ionization (ESI-) and longer retention times on a C18 column. This is well explained by a mechanistic model, which revealed that both polar and hydrophobic interactions are crucial for binding affinities. Encouraged by this, we then developed an SECC method to identify hL-FABP ligands, and all eight high-affinity ligands were selectively captured from 74 PFASs. The method was further applied to an aqueous film-forming foam (AFFF) product in which 31 new hL-FABP ligands were identified. Suspect and nontargeted screening revealed these ligands as analogues of perfluorosulfonic acids and homologues of alkyl ether sulfates (C8- and C10/EOn, C8H17(C2H4O)nSO4-, and C10H21(C2H4O)nSO4-). The SECC method was then applied to AFFF-contaminated surface waters. In addition to perfluorooctanesulfonic acid and perfluorohexanesulfonic acid, eight other AFFF chemicals were discovered as novel ligands, including four C14- and C15/EOn. This study implemented a high-throughput method to prioritize PFASs and revealed the existence of many previously unknown hL-FABP ligands.

Effect of polyethylene microplastics on tebuconazole bioaccumulation, oxidative stress, and intestinal bacterial community in earthworms.

Polyethylene microplastics (PE-MPs) are commonly found alongside fungicides in farmland soils. However, the toxic effects of varying PE-MP sizes and concentrations on soil fauna in fungicide-contaminated soils are unclear. This study aimed to investigate the impact of different PE-MP sizes (13, 48, and 150 µm) and concentrations (0.05 % and 0.25 %) on tebuconazole accumulation, oxidative stress, and gut bacteria in earthworms. The results indicated that earthworms exposed to MP13-H accumulated the highest tebuconazole on day 7, 19.77 % higher than tebuconazole alone, 7.27 % higher than MP48-H, and 10.30 % higher than MP150-H. MP13-H led to the most severe oxidative stress, significantly increasing the oxidative biomarkers catalase and peroxidase in earthworms. After 28 days, the expression of glutathione sulfotransferase genes was the lowest in the MP13-H group, while the antibacterial defense gene heat shock protein 70 and translationally controlled tumor protein were the highest, indicating severe DNA damage and increased toxicity to earthworms. Further, 150-µm PE-MPs caused the most severe damage to the intestinal epithelium. Moreover, PE-MPs induced an increase in the abundance of specific gut bacterial community associated with oxidative stress. The study suggested that PE-MPs changed the migration of fungicides to earthworms, induced oxidative stress, altered gene expression, and modified the gut microbiota, thereby increasing the risk to earthworms.

Emerging investigator series: nontargeted screening of aryl hydrocarbon receptor agonists in endangered beluga whales from the St. Lawrence Estuary: beyond legacy contaminants.

The elevated concentrations of organohalogen contaminants in the endangered St. Lawrence Estuary (SLE) belugas have prompted the hypothesis that aryl hydrocarbon receptor (AhR) activity may be a contributor towards their potential adverse effects. While indirect associations between AhR and contaminant levels have been reported in SLE beluga tissues, AhR activity was never directly measured. Using bioassays and nontargeted analysis, this study contrasted AhR activity and agonist profiles between pooled tissue extracts of endangered SLE and non-threatened Arctic belugas. Tissue extracts of SLE belugas exhibited significantly higher overall AhR activity than that of Arctic belugas, with a 2000s SLE beluga liver extract exerting significantly higher activity than blubber extracts of SLE and Arctic belugas from the same time period. Contrary to our expectations, well-known AhR agonists detected by nontargeted analysis, including polychlorinated biphenyls (PCBs), were only minor contributors to the observed AhR activity. Instead, Tox21 suspect screening identified more polar chemicals, such as dyes and natural indoles, as potential contributors. Notably, the natural product bromoindole was selectively detected in SLE beluga liver at high abundance and was further confirmed as an AhR agonist. These findings highlighted the significance of the AhR-mediated toxicity pathway in belugas and underscored the importance of novel AhR agonists, particularly polar compounds, in its induction.

Declining concentrations of chlorinated paraffins in endangered St. Lawrence Estuary belugas (Delphinapterus leucas): Response to regulations or a change in diet?

Very high levels of industrial contaminants in St. Lawrence Estuary (SLE) beluga whales represent one of the major threats to this population classified as endangered under the Species at Risk Act in Canada. Elevated concentrations of short-chained chlorinated paraffins (SCCPs) were recently reported in blubber of adult male SLE belugas. Recent regulations for SCCPs in North America, combined with their replacement by medium- (MCCPs) and long-chained chlorinated paraffins (LCCPs), highlight the importance of tracking this toxic chemical class. The objectives of this study were to evaluate (1) levels and profiles of chlorinated paraffins (CPs) in samples obtained from carcasses of adult male, adult female, juvenile, newborn, and fetus beluga, and (2) trends in adult male belugas between 1997 and 2018. Factors potentially influencing CP temporal trends such as age, feeding ecology and sampling year were also explored. SCCPs dominated (64 to 100%) total CP concentrations across all age and sex classes, MCCPs accounted for the remaining proportion of total CPs, and LCCPs were not detected in any sample. The chlorinated paraffin homolog that dominated the most in beluga blubber was C12Cl8. Adult male SCCP concentrations from this study were considerably lower (> 2000-fold) than those recently reported in Simond et al. (2020), likely reflecting a previously erroneous overestimate due to the lack of a suitable analytical method for SCCPs at the time. Both SCCPs and total CPs declined over time in adult males in our study (rate of 1.67 and 1.33% per year, respectively), presumably due in part to the implementation of regulations in 2012. However, there is a need to better understand the possible contribution of a changing diet to contaminant exposure, as stable isotopic ratios of carbon also changed over time.

Per- and Polyfluoroalkyl Substances in Canadian Fast Food Packaging.

A suite of analytical techniques was used to obtain a comprehensive picture of per- and polyfluoroalkyl substances (PFAS) in selected Canadian food packaging used for fast foods (n = 42). Particle-induced gamma ray emission spectroscopy revealed that 55% of the samples contained <3580, 19% contained 3580-10 800, and 26% > 10 800 µg F/m2. The highest total F (1 010 000-1 300 000 µg F/m2) was measured in molded "compostable" bowls. Targeted analysis of 8 samples with high total F revealed 4-15 individual PFAS in each sample, with 6:2 fluorotelomer methacrylate (FTMAc) and 6:2 fluorotelomer alcohol (FTOH) typically dominating. Up to 34% of the total fluorine was released from samples after hydrolysis, indicating the presence of unknown precursors. Nontargeted analysis detected 22 PFAS from 6 different groups, including degradation products of FTOH. Results indicate the use of side-chain fluorinated polymers and suggest that these products can release short-chain compounds that ultimately can be transformed to compounds of toxicological concern. Analysis after 2 years of storage showed overall decreases in PFAS consistent with the loss of volatile compounds such as 6:2 FTMAc and FTOH. The use of PFAS in food packaging such as "compostable" bowls represents a regrettable substitution of single-use plastic food packaging.

Widespread formation of toxic nitrated bisphenols indoors by heterogeneous reactions with HONO.

With numerous structurally diverse indoor contaminants, indoor transformation chemistry has been largely unexplored. Here, by integrating protein affinity purification and nontargeted mass spectrometry analysis (PUCA), we identified a substantial class of previously unrecognized indoor transformation products formed through gas-surface reactions with nitrous acid (HONO). Through the PUCA, we identified a noncommercial compound, nitrated bisphenol A (BPA), from house dust extracts strongly binding to estrogen-related receptor γ. The compound was detected in 28 of 31 house dust samples with comparable concentrations (ND to 0.30 µg/g) to BPA. Via exposing gaseous HONO to surface-bound BPA, we demonstrated it likely forms via a heterogeneous indoor chemical transformation that is highly selective toward bisphenols with electron-rich aromatic rings. We used 15N-nitrite for in situ labeling and found 110 nitration products formed from indoor contaminants with distinct aromatic moieties. This study demonstrates a previously unidentified class of chemical reactions involving indoor HONO, which should be incorporated into the risk evaluation of indoor contaminants, particularly bisphenols.

A Roadmap to the Structure-Related Metabolism Pathways of Per- and Polyfluoroalkyl Substances in the Early Life Stages of Zebrafish (Danio rerio).

BACKGROUND: Thousands of per- and polyfluoroalkyl substances (PFAS) with diverse structures have been detected in the ambient environment. Apart from a few well-studied PFAS, the structure-related toxicokinetics of a broader set of PFAS remain unclear. OBJECTIVES: To understand the toxicokinetics of PFAS, we attempted to characterize the metabolism pathways of 74 structurally diverse PFAS samples from the U.S. Environmental Protection Agency's PFAS screening library. METHODS: Using the early life stages of zebrafish (Danio rerio) as a model, we determined the bioconcentration factors and phenotypic toxicities of 74 PFAS. Then, we applied high-resolution mass spectrometry-based nontargeted analysis to identify metabolites of PFAS in zebrafish larvae after 5 d of exposure by incorporating retention time and mass spectra. In vitro enzymatic activity experiments with human recombinant liver carboxylesterase (hCES1) were employed to validate the structure-related hydrolysis of 11 selected PFAS. RESULTS: Our findings identified five structural categories of PFAS prone to metabolism. The metabolism pathways of PFAS were highly related to their structures as exemplified by fluorotelomer alcohols that the predominance of ß-oxidation or taurine conjugation pathways were primarily determined by the number of hydrocarbons. Hydrolysis was identified as a major metabolism pathway for diverse PFAS, and perfluoroalkyl carboxamides showed the highest in vivo hydrolysis rates, followed by carboxyesters and sulfonamides. The hydrolysis of PFAS was verified with recombinant hCES1, with strong substrate preferences toward perfluoroalkyl carboxamides. CONCLUSIONS: We suggest that the roadmap of the structure-related metabolism pathways of PFAS established in this study would provide a starting point to inform the potential health risks of other PFAS. https://doi.org/10.1289/EHP7169.