Abstract-Perfluorooctane sulfonic acid (PFOS) accumulates in the liver and blood of exposed organisms. The potential for these surfactant molecules to interfere with hormone/protein interactions in blood is of concern given the importance of these interactions. The PFOS binding to serum proteins was investigated by assessing its ability to displace a variety of steroid hormones from specific binding proteins in the serum of birds and fishes. Perfluorooctane sulfonic acid had only a weak ability to displace estrogen or testosterone from carp serum steroid binding proteins. Displacement of cortisone in avian sera occurred at relatively low PFOS concentrations. Corticosterone displacement potency increased with chain length, and sulfonic acids were more potent than carboxylic acids. The PFOS concentrations estimated to cause these effects were 320 M or greater, equivalent to serum concentrations greater than 160 mg/L. Using mass spectrometry and direct in vitro binding assays, PFOS was demonstrated to bind strongly to bovine serum albumin (BSA) in a 1:1 stoichiometric ratio. It appears that PFOS in serum is in general bound to albumins. Concentrations of PFOS required to saturate albumin would be in excess of 50 to 100 mg/L. Based on current environmental concentrations, it is unlikely that PFOS would cause displacement of hormones from serum proteins in wildlife.

Perfluorooctane sulfonate (PFOS) is a perfluorinated molecule that has recently been identified in the sera of nonindustrially exposed humans. In this study, 247 tissue samples from 15 species of marine mammals collected from Florida, California, and Alaskan coastal waters; and northern Baltic Sea; the Arctic (Spitsbergen); and Sable Island in Canada were analyzed for PFOS. PFOS was detected in liver and blood of marine mammals from most locations including those from Arctic waters. The greatest concentrations of PFOS found in liver and blood were 1520 ng/g wet wt in a bottlenose dolphin from Sarasota Bay, FL, and 475 ng/mL in a ringed seal from the northern Baltic Sea (Bothnian Sea), respectively. No age-dependent increase in PFOS concentrations in marine mammals was observed in the samples analyzed. The occurrence of PFOS in marine mammals from the Arctic waters suggests widespread global distribution of PFOS including remote locations.

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants. The structurally related hydroxylated PBDEs (OH-PBDEs) and methoxylated PBDEs (MeO-PBDEs) occur in precipitation, surface water, wildlife, and humans. The formation of OH-PBDEs in wildlife and humans is of considerable concern due to their greater toxicities relative to PBDEs and MeO-PBDEs. Research to date suggests that OH-PBDEs are formed by hydroxylation of PBDEs, and MeO-PBDEs are then formed by methylation of the OH-PBDEs. Here we show significant metabolic production of OH-PBDEs from MeO-PBDEs while hydroxylation of synthetic PBDEs to OH-PBDEs was negligible. Concentrations of PBDEs, OH-PBDEs, and MeO-PBDEs were analyzed in tuna, albatross, and polar bears collected from marine environments worldwide, and we found a closer relationship between OH-PBDEs and MeO-PBDEs than had been previously reported. Furthermore, for the first time the metabolic relationships between PBDEs, OH-PBDEs, and MeO-PBDEs were elucidated in vitro using rainbow trout, chicken, and rat microsomes. We propose the production of OH-PBDEs from naturally occurring MeO-PBDEs as a previously unidentified mechanism that could be an important contributor for the occurrence of OH-PBDEs found in wildlife from remote areas. Our results suggest that risk assessment paradigms for PBDEs and their metabolites need reevaluation and that human exposure to MeO-PBDEs that occur naturally in marine organisms should be considered.

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent α-cyanoester and α-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxynaphthalen-2-yl)methyl trans- [(3-phenyloxiran-2-yl) methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC 50 values for a series of known inhibitors. The new IC 50 values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC 50 values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay. KeywordsSoluble epoxide hydrolase; α-Cyanoester; α-Cyanocarbonate; Kinetic assay; Fluorescent substrateThe soluble epoxide hydrolase (sEH, EC 3.3.2.3) 1 is a member of the α/β-hydrolase fold family of enzymes [1] and catalyzes the hydrolysis of an epoxide to its corresponding diol through the catalytic addition of a water molecule [2]. The endogenous substrates for the sEH include epoxides of arachidonic acid [3,4] and linoleic acid [5,6]. Arachidonic acid epoxides (epoxyeicosatrienoic acid epoxides, EETs) are known modulators of blood pressure [7,8] and vascular permeability [9,10]. It has been shown that sEH inhibition not only lowers blood * Corresponding author. Fax: +1 530 752 1537. E-mail address:bdhammock@ucdavis.edu (B.D. Hammock).. 1 Abbreviations used: sEH, soluble epoxide hydrolase; EET, epoxyeicosatrienoic acid epoxides; NEPC, 4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate; TEA, triethylamine; TLC, thin-layer chromatography; TMS, tetramethylsilane; ppm, parts per million; oa-TOF, orthogonal acceleration time-of-flight; THF, tetrahydrofuran; m-CPBA, m-chloroperbenzoic acid; BSA, bovine serum albumin; RFU, relative fluorescent units; OD, optical density; DMSO, dimethyl sulfoxide; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; tDPPO, [ 3 H] pressure in rodent models but also offers protection against hypertension-related renal damage [4,11,12].For the past 6 years, we have investi...

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During inflammation, a large amount of arachidonic acid (AA) is released into the cellular milieu and cyclooxygenase enzymes convert this AA to prostaglandins that in turn sensitize pain pathways. However, AA is also converted to natural epoxyeicosatrienoic acids (EETs) by cytochrome P450 enzymes. EET levels are typically regulated by soluble epoxide hydrolase (sEH), the major enzyme degrading EETs. Here we demonstrate that EETs or inhibition of sEH lead to antihyperalgesia by at least 2 spinal mechanisms, first by repressing the induction of the COX2 gene and second by rapidly up-regulating an acute neurosteroid-producing gene, StARD1, which requires the synchronized presence of elevated cAMP and EET levels. The analgesic activities of neurosteroids are well known; however, here we describe a clear course toward augmenting the levels of these molecules. Redirecting the flow of pronociceptive intracellular cAMP toward up-regulation of StARD1 mRNA by concomitantly elevating EETs is a novel path to accomplish pain relief in both inflammatory and neuropathic pain states.cAMP ͉ inflammatory pain ͉ steroidogenesis

A series of 1,3-disubstituted ureas possessing a piperidyl moiety has been synthesized to investigate their structure-activity relationships as inhibitors of the human and murine soluble epoxide hydrolase (sEH). Oral administration of thirteen 1-aryl-3-(1-acylpiperidin-4-yl)urea inhibitors in mice revealed substantial improvements in pharmacokinetic parameters over previously reported 1-adamantyl-urea based inhibitors. For example, 1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (52) showed a 7-fold increase in potency, a 65-fold increase in Cmax† and a 3300 fold increase in AUC over its adamantane analogue 1-(1-adamantyl)-3-(1-propionylpiperidin-4-yl)urea (2). This novel sEH inhibitor showed a 1000 fold increase in potency when compared to morphine by reducing hyperalgesia as measured by mechanical withdrawl threshold using the in vivo carrageenan induced inflammatory pain model.

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