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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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Diosbulbin B (DIOB), a furan-containing diterpenoid lactone, is the most abundant component of Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Administration of purified DIOB or DB extracts has been reported to cause liver injury in animals. The mechanisms of DIOB-induced hepatotoxicity remain unknown. The major objective of this study was to identify reactive metabolites of DIOB. A DIOB-derived cis-enedial was trapped by N-acetyl lysine (NAL) and glutathione (GSH) or N-acetyl cysteine (NAC) in rat and human liver microsomal incubation systems after exposure to DIOB. Four metabolites (M1-M4) associated with GSH were detected by liquid chromatography coupled to tandem mass spectrometry. Apparently, M1 was derived from both NAL and GSH. M2 and M3 resulted from the reaction of GSH without the involvement of NAL. Two molecules of GSH participated in the formation of M4. M2 and M3 were also detected in bile and urine of rats given DIOB. M5, a DIOB-derived NAC/NAL conjugate, was detected in microsomal incubations with DIOB fortified with NAC and NAL as trapping agents. Biomimetic M1-M5 were prepared by oxidation of DIOB with Oxone for metabolite identification. Microsomal incubation study demonstrated that ketoconazole inhibited the production of the enedial in a concentration-dependent manner, and CYP3A4 was found to be the enzyme responsible for the metabolic activation of DIOB. The metabolism study facilitates the understanding of the role of bioactivation of DIOB in its hepatotoxicity.

ABSTRACT:Clivorine, a naturally occurring pyrrolizidine alkaloid, causes liver toxicity via its metabolic activation to generate toxic metabolite (pyrrolic ester). Female Sprague-Dawley (SD) rats are reported to be less susceptible to clivorine intoxication than male SD rats. However, the biochemical mechanism causing such gender difference is largely unknown. The present study investigated hepatic microsomal metabolism of clivorine in female rats to delineate the mechanism of the gender difference. Two pathways, which directly metabolize clivorine, were observed. First, the metabolic activation to produce the toxic pyrrolic ester followed by formations of bound pyrroles, dehydroretronecine, 7-glutathionyldehydroretronecine, and clivoric acid were found in female rats, and CYP3A1/2 isozymes were identified to catalyze the metabolic activation. Compared with male rats (ϳ21%), the metabolic activation in female rats was significantly lower (ϳ4%) possibly because of significantly lower CYP3A1/2 levels expressed in female rats. Second, a direct hydrolysis to generate the novel female rat-specific metabolite deacetylclivorine was shown as the predominant pathway (ϳ16% clivorine metabolism) in female rat liver microsomes and was determined to be mediated by microsomal hydrolase A. Furthermore, when the metabolic activation was completely inhibited by ketoconazole, the amount of deacetylclivorine formed in a 1-h incubation significantly increased from 19.44 ؎ 3.00 to 54.87 ؎ 9.30 nmol/mg protein, suggesting that the two pathways compete with each other. Therefore, the lower susceptibility of female SD rats to clivorine intoxication is suggested to be caused by the significantly higher extent of the direct hydrolysis and a lower degree of the metabolic activation.Pyrrolizidine alkaloid (PA) poisoning has drawn worldwide attention because of a wide distribution of PA-containing plants and their induced serious and diversified toxicities, especially hepatotoxicity and carcinogenicity (Mattocks, 1968;Mori et al., 1985;Huxtable, 1989;Buhler et al., 1990;Fu et al., 2002Fu et al., , 2004, as well as pneumotoxicity (Huxtable, 1990;Taylor et al., 1997), neurotoxicity (Roeder, 2000), and embryotoxicity (Tu et al., 1988). Two types of PA, namely, retronecine and otonecine, are mainly responsible for the PA-induced hepatotoxicity (Mori et al., 1985;Huxtable, 1989;Buhler et al., 1990;Fu et al., 2004). Clivorine, a representative toxic otonecine-type PA, is present in many Ligularia species and especially exists as a predominant PA in the traditional Chinese medicinal herb Ligularia hodgsonii Hook (Lin et al., 2000b;Xia et al., 2004). Clivorine has been reported to cause hepatotoxicity and carcinogenicity in rodents and a positive mutagenic response in the Ames test in the presence of rat liver homogenates, suggesting the importance of hepatic metabolic activation in its intoxication (Yamanaka et al., 1979;Kuhara et al., 1980;Xia et al., 2004). In our previous studies, hepatic microsomal metabolism of clivorine in male Sprague-Dawle...

Pyrrolizidine alkaloids (PAs) induce liver injury (PA-ILI) and is very likely to contribute significantly to drug-induced liver injury (DILI). In this study we used a newly developed ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-MS)-based method to detect and quantitate blood pyrrole-protein adducts in DILI patients. Among the 46 suspected DILI patients, 15 were identified as PA-ILI by the identification of PA-containing herbs exposed. Blood pyrrole-protein adducts were detected in all PA-ILI patients (100%). These results confirm that PA-ILI is one of the major causes of DILI and that blood pyrrole-protein adducts quantitated by the newly developed UHPLC-MS method can serve as a specific biomarker of PA-ILI.

Diosbulbin B (DIOB), a furanoid, is a major constituent of herbal medicine Dioscorea bulbifera L. Exposure to DIOB caused liver injury in humans and experimental animals. The mechanisms of DIOB-induced hepatotoxicities remain unknown. The present study demonstrated that DIOB induced hepatotoxicities in a time- and dose-dependent manner in mice. H&E stained histopathologic image showed the occurrence of necrosis in the liver obtained from the mice treated with DIOB at dose of 200 mg/kg. Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB, increased area under the concentration-time curve of blood DIOB, decreased urinary excretion of GSH conjugates derived from DIOB, and increased urinary excretion of parent drug. Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities. In order to define the role of furan moiety in DIOB-induced liver toxicities, we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB. No liver injury was observed in the animals given the same doses of tetrahydro-DIOB. The furan moiety was essential for DIOB-induced hepatotoxicities. The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities. The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites, and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules. Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.

Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.