The human fecal microbiome contributes to the biotransformation of the PFAS surfactant 8:2 monosubstituted polyfluoroalkyl phosphate ester.

Polyfluoroalkyl phosphate esters (PAPs) can be found throughout society due to their numerous commercial applications. However, they also pose an environmental and health concern given their ability to undergo hydrolysis and oxidation to several bioactive and persistent products, including the perfluorocarboxylic acids (PFCAs). The metabolism of PAPs has been shown to occur in mammalian liver and intestine, however metabolism by the gut microbiome has not yet been investigated. In this study, human fecal samples were used to model the microbial population of the colon, to test whether these anaerobic microbes could facilitate 8:2 monosubstituted PAP (monoPAP) transformation. In vitro testing was completed by incubating the fecal samples with 8:2 monoPAP (400-10,000 nM) up to 120 minutes in an anaerobic chamber. Reactions were then terminated and the samples prepared for GC- and LC-MS/MS analysis. Metabolites of interest were the immediate hydrolysis product, the 8:2 fluorotelomer alcohol (FTOH), and 11 additional metabolites previously shown to form from 8:2 FTOH in both oxic and anoxic environments. The kinetics of 8:2 monoPAP transformation by gut microbiota were compared to those in human S9 liver and intestine fractions, both of which have active levels of hydrolyzing and oxidative enzymes that transform 8:2 monoPAP. Transformation rates from 8:2 monoPAP to 8:2 FTOH were highest in liver S9 > intestine S9 > fecal suspensions. The gut microbiome also produced a unique composition of oxidative metabolites, where the following intermediate metabolites were more abundant than terminal PFCAs: 8:2 fluorotelomer unsaturated carboxylic acid (FTUCA) > 8:2 fluorotelomer carboxylic acid (FTCA) > 7:2 Ketone ≈ perfluorohexanoic acid (PFHxA). Hydrolytic and oxidative metabolites contributed up to 30% of the molar balance after microbial 8:2 monoPAP transformation. Together, the results suggest that the gut microbiome can play a notable role in PAP biotransformation.

Emerging investigator series: human CYP2A6 catalyzes the oxidation of 6:2 fluorotelomer alcohol.

The biotransformation of 6:2 fluorotelomer alcohol (6:2 FTOH) results in the production of bioactive and persistent metabolites, including perfluorinated carboxylic acids (PFCAs). While the products of 6:2 FTOH metabolism have been elucidated in several animal models, the responsible cytochrome P450 (CYP) isoform(s) have not been reported. Here, we characterized the in vitro oxidation of 6:2 FTOH using human liver microsomes and recombinant human CYPs. Six major xenobiotic metabolizing CYPs were screened for their capacity to catalyze 6:2 FTOH oxidation using chemical inhibitors selective towards CYP isoforms. Of the CYP isoforms investigated, CYP2A6 was the only enzyme capable of catalyzing 6:2 FTOH in human liver microsomes, with KM and Vmax values of 4076 ng mL-1 and 69 ng mL-1 min-1, respectively. We further probed the metabolic mechanism by plotting the 6:2 FTOH kinetic profile and extrapolating data to several possible kinetic models. 6:2 FTOH oxidation followed the typical one-site Michaelis-Menten kinetic model. This study also reports that 6:2 FTOH loss is associated with active CYP2A6 by incubating microsomes with the selective CYP2A6 inhibitor tranylcypromine, which bound competitively to the enzyme as determined by an increased KM (8796 ng mL-1) but unchanged Vmax value. Collectively, these findings provide a mechanistic perspective on the potential importance of CYP2A6 in the metabolic activation and phase I elimination of 6:2 FTOH and indirect human exposure to PFCAs.

Epoxyeicosatrienoic acid (EET)-stimulated angiogenesis is mediated by epoxy hydroxyeicosatrienoic acids (EHETs) formed from COX-2.

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

Cyclooxygenase-derived proangiogenic metabolites of epoxyeicosatrienoic acids.

Arachidonic acid (ARA) is metabolized by cyclooxygenase (COX) and cytochrome P450 to produce proangiogenic metabolites. Specifically, epoxyeicosatrienoic acids (EETs) produced from the P450 pathway are angiogenic, inducing cancer tumor growth. A previous study showed that inhibiting soluble epoxide hydrolase (sEH) increased EET concentration and mildly promoted tumor growth. However, inhibiting both sEH and COX led to a dramatic decrease in tumor growth, suggesting that the contribution of EETs to angiogenesis and subsequent tumor growth may be attributed to downstream metabolites formed by COX. This study explores the fate of EETs with COX, the angiogenic activity of the primary metabolites formed, and their subsequent hydrolysis by sEH and microsomal EH. Three EET regioisomers were found to be substrates for COX, based on oxygen consumption and product formation. EET substrate preference for both COX-1 and COX-2 were estimated as 8,9-EET > 5,6-EET > 11,12-EET, whereas 14,15-EET was inactive. The structure of two major products formed from 8,9-EET in this COX pathway were confirmed by chemical synthesis: ct-8,9-epoxy-11-hydroxy-eicosatrienoic acid (ct-8,9-E-11-HET) and ct-8,9-epoxy-15-hydroxy-eicosatrienoic acid (ct-8,9-E-15-HET). ct-8,9-E-11-HET and ct-8,9-E-15-HET are further metabolized by sEH, with ct-8,9-E-11-HET being hydrolyzed much more slowly. Using an s.c. Matrigel assay, we showed that ct-8,9-E-11-HET is proangiogenic, whereas ct-8,9-E-15-HET is not active. This study identifies a functional link between EETs and COX and identifies ct-8,9-E-11-HET as an angiogenic lipid, suggesting a physiological role for COX metabolites of EETs.

Is there a human health risk associated with indirect exposure to perfluoroalkyl carboxylates (PFCAs)?

The production and widespread use of poly- and perfluoroalkyl substances (PFAS) has led to their presence in the environment, wildlife, and humans. Particularly, the perfluoroalkyl carboxylates (PFCAs) are pervasive throughout the world and have been found at ng/mL concentrations in human blood. PFCAs, especially those having longer carbon chain lengths (≥C6), are associated with developmental and hormonal effects, immunotoxicity, and promote tumor growth in rodents through their role as PPARα agonists. Humans are directly exposed to PFCAs primarily through contaminated food, drinking water, and house dust. However, indirect exposure to PFCAs through the biotransformation of fluorotelomer-based substances may also be a significant, yet relatively underappreciated pathway. We are exposed to fluorotelomer-based substances through use of consumer products, ingestion of food, or from inhalation of dust particles, but the risk of this exposure has been largely uncharacterized. Here, we summarize the work that has been done to characterize toxicity of the classes of fluorotelomer-based substances shown to biotransform to PFCAs: the polyfluoroalkyl phosphate esters (PAPs), fluorotelomer alcohols (FTOHs), fluorotelomer iodides (FTIs), and fluorotelomer acrylate monomers (FTAcs). These fluorotelomer-based substances biotranform to yield PFCAs, yet also form bioactive intermediate metabolites, which have been observed to be more toxic than their corresponding PFCAs. We address what is known regarding the toxicity of the fluorotelomer-based substances and their metabolites, with focus on covalent binding to biological nucleophiles, such as glutathione, proteins, and DNA, as a possible mechanism of toxicity that may influence the risk of indirect exposure to PFCAs.

In vitro interactions of biological nucleophiles with fluorotelomer unsaturated acids and aldehydes: fate and consequences.

Fluorotelomer unsaturated aldehydes and acids (FTUALs and FTUCAs) are intermediate metabolites that form from the biotransformation of fluorotelomer-based chemicals. FTUALs and FTUCAs have been previously suggested to contribute to the toxicity associated with human exposure to fluorotelomer compounds by covalently binding to biological nucleophiles. However, the extent of their reactivity has only been assessed with glutathione. The purpose of the present study was to assess the reactivity of these intermediate metabolites with a series of nucleophilic amino acids and model proteins. In vitro experiments were carried out in an aqueous buffer system to determine the reactivity of nucleophilic amino acids with FTUCAs and FTUALs having varying fluorinated chain lengths. Using (19)F NMR spectroscopy to monitor the disappearance of the FTUCAs and FTUAL signals and the production of a fluoride signal, reaction rate constants were determined under pseudo-first-order conditions. The FTUCAs reacted only with cysteine with the following second order rate constants: 3.63 (± 1.37) × 10(-5) min(-1) mM(-1) (4:2 FTUCA), 1.19 (± 0.91) × 10(-5) min(-1) mM(-1) (6:2 FTUCA), and 4.56 (± 0.94) × 10(-5) min(-1) mM(-1) (8:2 FTUCA). The FTUALs were significantly more reactive than any of the FTUCAs with reactivity decreasing in the following order: cysteine >> histidine > lysine >> arginine. The following second-order rate constants were obtained: 5.7 (± 4.2) × 10(-4) min(-1) mM(-1) (histidine), 4.3 (± 1.4) × 10(-4) min(-1) mM(-1) (lysine), and 1.4 (± 0.73) × 10(-4) min(-1) mM(-1) (arginine). FTUCAs and FTUALs were also reacted with model proteins to assess their potential for forming covalent adducts. Electrospray ionization mass spectrometry (ESI-MS) was used to investigate the stoichiometry of FTUCAs and FTUALs covalently bound to apomyoglobin (ApoMg) and human serum albumin (HSA). FTUCAs were not reactive, whereas two measurable FTUAL adducts were formed with both ApoMg and HSA at each of the FTUAL chain lengths (6:2, 8:2, and 10:2). This is the first study to probe the reactivity of FTUALs and FTUCAs with nucleophiles other than glutathione, further elucidating possible FTUAL and FTUCA fate within biological systems.

Assessing the structure-activity relationships of fluorotelomer unsaturated acids and aldehydes with glutathione. Reactivity of glutathione with fluorotelomer unsaturated acids and aldehydes.

Fluorotelomer alcohols (FTOHs) have been shown to degrade via abiotic and biotic mechanisms to perfluorocarboxylates (PFCAs) which are environmentally persistent and bioaccumulate in humans and wildlife depending on their chain length. Fluorotelomer unsaturated aldehydes (FTUALs) and acids (FTUCAs) are intermediate metabolites that form from the degradation of FTOHs. Their potential for toxicity is not yet defined and may be more significant compared to PFCAs. Past studies have shown that these intermediates form adducts with glutathione (GSH). The purpose of this study was to further assess the reactivity of these intermediate compounds. In vitro experiments were carried out in an aqueous buffer system (pH 7.4) where FTUCAs and FTUALs of varying chain lengths were reacted with GSH. To quantify the reactivity of FTUCAs and FTUALs, unreacted free GSH was derivatized with 5,5'-dithiobis(2-nitrobenzoic acid), its absorbance measured at 412 nm, and the percentage of unconjugated free GSH evaluated over time. EC50 values were obtained for the reactions of GSH with acrolein and methyl methacrylate to assess the accuracy of the method, as well as for acrylic acid, FTUCAs, and FTUALs. The results of this study indicated that α,ß-unsaturated aldehydes are comparatively the most reactive and reaction with GSH may be influenced by the length of the fluorinated tail. This is the first study to examine the relationship of FTUCAs and FTUALs with biological nucleophiles by quantifying their intrinsic reactivity.

Polypyrroles as antioxidants: kinetic studies on reactions of bilirubin and biliverdin dimethyl esters and synthetic model compounds with peroxyl radicals in solution. Chemical calculations on selected typical structures.

[reaction: see text] Rate constants for hydrogen-atom transfer (HAT) from bilirubin dimethyl ester (BRDE) and biliverdin dimethyl ester (BVDE) to peroxyl radicals during inhibited autoxidation of styrene initiated by azo-bisisobutyronitrile (AIBN) were k(inh)(BRDE) = 22.5 x 10(4) and k(inh)(BVDE) = 10.2 x 10(4) M(-1) s(-1), and the stoichiometric factors (n) were 2.0 and 2.7, respectively. A synthetic tetrapyrrole (bis(dipyrromethene)) containing the alpha-central (2,2') CH2 linkage gave k(inh) = 39.9 x 10(4) M(-1) s(-1) and n = 2.3, whereas the beta-linked (3,3') isomer was not an active antioxidant. Several dipyrrinones were synthesized as mimics of the two outer heterocyclic rings of bilirubin and biliverdin. The dipyrrinones containing N-H groups in each ring were active antioxidants, whereas those lacking two such "free" N-H groups, such as N-CH3 dipyrrinones and dipyrromethenes, did not exhibit antioxidant activity. Overall, the relative k(inh) values compared to those of phenolic antioxidants, 2,6-di-tert-butyl-4-methoxyphenol (DBHA) and 2,6-di-tert-butyl-4-methylphenol (BHT), were 2,2'-bis(dipyrromethene) > BRDE > DBHA > dipyrrinones > BVDE > BHT. This general trend in antioxidant activities was also observed for the inhibited autoxidation of cumene initiated by AIBN. Chemical calculations of the N-H bond dissociation enthalpies (BDEs) of the typical structures support a HAT mechanism from N-H groups to trap peroxyl radicals. Intramolecular hydrogen bonding of intermediate nitrogen radicals has a major influence on the antioxidant activities of all compounds studied. Indeed, chemical calculations showed that the initial nitrogen radical from a dipyrrinone is stabilized by 9.0 kcal/mol because of H-bonding between the N-H remaining on one ring and the ground-state pyrrolyl radical of the adjacent ring in the natural zusammen structure. The calculated minimum structure of bilirubin shows strong intramolecular H-bonding of the N-H groups with carbonyl groups resulting in the known "ridge-tile" structure which is not an active HAT antioxidant. The calculated minimum structure of biliverdin is planar. BRDE is readily converted into BVDE by reaction with the electron-deficient DPPH* radical under argon in chlorobenzene. An electron-transfer mechanism is proposed for the initiating step in this reaction, and this is supported by the relatively low ionizing potential of a model dipyrrole representing the two central rings of bilirubin.