The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inf lammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tightbinding inhibitors with nanomolar K i values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of transstilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.Epoxide hydrolases (EH; E.C.3.3.2.3) catalyze the hydrolysis of epoxides or arene oxides to their corresponding diols by the addition of water (1). In mammals, the hepatic microsomal and soluble epoxide hydrolase forms are known to complement each other in detoxifying a wide array of mutagenic, toxic, and carcinogenic, xenobiotic epoxides (2, 3). Soluble EH (sEH) is also involved in the metabolism of arachidonic (4) and linoleic (5) acid epoxides. Arachidonate epoxides and diols are elevated in association with pregnancy-induced hypertension and modulate vascular permeability in the heart and kidneys (6). Diols derived from epoxy-linoleate (leukotoxin) perturb membrane permeability and calcium homeostasis (5), resulting in inflammation modulated by nitric oxide synthase and endothelin-1 (7, 8). Micromolar concentrations of leukotoxin reported in association with inflammation and hypoxia (9) depress mitochondrial respiration in vitro (10) and cause mammalian cardiopulmonary toxicity in vivo (7,11,12). Leukotoxin toxicity presents symptoms suggestive of multiple organ failure and acute respiratory distress syndrome (9). In both cellular and organismal models, leukotoxin-mediated toxicity depends on epoxide hydrolysis (5).The bioactivity of these epoxide hydrolysis products and their association with inflammation suggest that inhibition of vicinal-dihydroxylipid biosynthesis may have therapeutic value, making sEH a promising pharmacological target. Previously described selective sEH inhibitors, substituted chalcone oxides (as compound 1 in Table 1), and phenylglycidols (13,14) are epoxides that are hydrolyzed slowly by the target enzyme. Inhibition stems from an electronically stabilized covalent intermediate that results in low turnover and transient inhibition (15). Moreover, these compounds are relatively unstable, particularly in the presence of glutathione (13), making them of limited use in vivo. We describe herein the discovery of new potent and stable inhibitors of soluble EH and their application to both in vitro and in vivo models. MATERIALS AND METHODSSynthesis. Compounds 2, 3, 7-9, and 11-18 were obtained from Aldri...

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The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods.

Two families of carboxylic acid derivatives of the herbicides atrazine and simazine were synthesized for use as haptens in the development of immunoassays. One family was made by using monosubstituted (alky1amino)cyanuric chlorides and a variety of w-amino acids, giving spacers of varying lengths attached to one secondary amino group. The second family resulted from the replacement of the 2-chloro group of atrazine or simazine with 3-mercaptopropanoic acid. These haptens were conjugated to carrier proteins via N-hydroxysuccinimide active esters to make immunogens and ELISA antigens. Mono(alky1amino)cyanuric chlorides were also conjugated directly to bovine serum albumin and thyroglobulin under physiological conditions. ELISA and competitive inhibition ELISA results demonstrated that antibodies from immunized rabbits bound all of the conjugated and free s-triazines tested, including the parent compounds atrazine and simazine. The degree of binding was dependent on the N-alkyl groups present and on the length and position of attachment of the spacer arm. These results illustrate the usefulness of this synthetic approach in easily producing immunogenic haptens for a class of compounds that are difficult to work with because of their low solubility.The s-triazine herbicides are among the most commonly detected pesticides in water (Muir et al., 1978; Glotfelty e t al., 1984;Ervin and Kittleson, 1988;LeMasters and Doyle, 1989). This observation is due to a combination of factors, including widespread and heavy use, chemical and biological stability, and mobility in water. From 1983 through 1987 over 3 000 000 pounds of simazine and atrazine (Figure 1; 3a and 3b) were used in California alone (California Department of Food and Agriculture, [1983][1984][1985][1986][1987]. For these reasons, the State of California considers them as prime indicator compounds of groundwater contamination. Of additional concern is the poorly understood potential of the s-triazines and their nitroso and other derivatives for teratogenic, mutagenic, and carcinogenic effects (Janzowski et al., 1980; Waters et al., 1981). Thus, a rapid and inexpensive assay is needed to allow rapid screening of numerous surface and subsurface water samples, as well as for a variety of other analytical applications. The value of immunoassays for the analysis of pesticide residues has been well established (Hammock and Mumma, 1980; Newsome, 1986; Harrison e t al., 1988; Van Emon et al., 1989; Jung e t al., 1989). Recent literature includes descriptions of immunoassays for three specific s-triazine herbicides, atrazine (Huber, 1985; Bushway et al., 1988;Dunbar et al., 1985;Schlaeppi et al., 1989), cyanazine (Robotti et al., 1986), and terbutryne (Huber and Hock, 1985).Since the commercially important s-triazine herbicides constitute a large class of structurally related compounds that should be amenable to immunoassay as a group, we chose a synthetic approach that allows for the development of a wider range of antibody specificities and ultimate assay designs tha...

Enzyme-linked immunosorbent assays (ELISAs) are reported for the detection of atrazine and its principle metabolite in human urine. The ELISAs can be used with crude urine or following extraction and partial purification by methods described in this report. GC, MS, and HPLC techniques were used to confirm and complement the ELISA methods for qualitative and quantitative detection of urinary metabolites. A series of samples from workers applying this herbicide confirmed a mercapturic acid conjugate of atrazine as a major urinary metabolite. The mercapturate was found in concentrations at least 10 times that of any of the N-dealkylated products or the parent compound. Atrazine mercapturic acid was isolated from urine using affinity extraction based upon a polyclonal antibody for hydroxy-s-triazines and yielded products sufficiently pure for structure confirmation by MS/MS. In a pilot study monitoring applicators, a relationship between cumulative dermal and inhalation exposure and total amount of atrazine equivalents excreted over a 10-day period was observed. On the basis of these data, we propose that an ELISA for the mercapturate of atrazine could be developed as a useful marker of exposure.