The telltale structures of epoxide hydrolases

Traditionally, epoxide hydrolases (EH) have been regarded as xenobiotic-metabolizing enzymes implicated in the detoxification of foreign compounds. They are known to play a key role in the control of potentially genotoxic epoxides that arise during metabolism of many lipophilic compounds. Although this is apparently the main function for the mammalian microsomal epoxide hydrolase (mEH), evidence is now accumulating that the mammalian soluble epoxide hydrolase (sEH), despite its proven role in xenobiotic metabolism, also has a central role in the formation and breakdown of physiological signaling molecules. In addition, a certain class of microbial epoxide hydrolases has recently been identified that is an integral part of a catabolic pathway, allowing the use of specific terpens as sole carbon sources. The recently available x-ray structures of a number of EHs mirror their respective functions: the microbial terpen EH differs in its fold from the canonical alpha/beta hydrolase fold of the xenobiotic-metabolizing mammalian EHs. It appears that the latter fold is the perfect solution for the efficient detoxification of a large variety of structurally different epoxides by a single enzyme, whereas the smaller microbial EH, which has a particularly high turnover number with its prefered substrate, seems to be the better solution for the hydrolysis of one specific substrate. The structure of the sEH also includes an additional catalytic domain that has recently been shown to possess phosphatase activity. Although the physiological substrate for this second active site has not been identified so far, the majority of known phosphatases are involved in signaling processes, suggesting that the sEH phosphatase domain also has a role in the regulation of physiological functions.     

Mammalian epoxide hydrolases in xenobiotic metabolism and signalling

Epoxide hydrolases catalyse the hydrolysis of electrophilic—and therefore potentially genotoxic—epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)—an enzyme prone to detoxification—due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structure.

Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is suggested to alter the mode of action and increase the toxic potency of fatty acid epoxides. To characterize the structural features necessary for sEH-dependent epoxy fatty acid toxicity, 75 aliphatic compounds were assayed for cytotoxicity in the presence and absence of sEH. Three groups of aliphatic epoxide-diol pairs were described by their observed differential toxicity. Group I compounds were typified by terminal epoxides whose toxicity was reduced in the presence of sEH. Group II compounds were toxic in either their epoxide or diol form, but toxicity was unaffected by sEH. Group III compounds exhibited sEH-dependent toxicity and were therefore used to investigate the structural elements required for cytotoxicity in this study. The optimal structure for group III compounds appeared to be a fatty acid 18-20 atoms long (e.g., a carbon backbone plus a terminal heteroatom) with an epoxide positioned between C-7 and C-12. In the absence of sEH, replacement of epoxides with a vicinal diol was required for toxicity. While diol stereochemistry was unimportant, vicinal diol-induced toxicity exhibited fewer positional constraints to toxicity than sEH-dependent epoxide toxicity. Tested fatty acids and esters with neither an epoxide nor a vicinal diol were not toxic. These data support the hypothesis that long-chain epoxy fatty acid methyl esters are potential pro-toxins metabolized by sEH to more toxic diols. Furthermore, our results suggest that the endogenous compounds, leukotoxin methyl ester, 9,10(Z)-epoxyoctadec-12(Z)-enoic acid methyl ester, and isoleukotoxin methyl ester, 12, 13(Z)-epoxyoctadec-9(Z)-enoic acid methyl ester, are structurally optimized to elicit the observed effect.     

Inhibition of soluble and microsomal epoxide hydrolase by zinc and other metals.

Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.     

Discovery of soluble epoxide hydrolase inhibitors from natural products

With the goal of developing soluble epoxide hydrolase (sEH) inhibitors with novel chemical structures, the sEH inhibitory activities of 30 natural compounds were evaluated using both a fluorescent substrate, 3-phenyl-cyano(6-methoxy-2-naphthalenyl)methyl ester- 2-oxiraneacetic acid, and a physiological substrate, 14,15-epoxyeicosatrienoic acid. To evaluate the selectivity of sEH inhibition, the inhibition of microsomal epoxide hydrolase (mEH), which plays a critical role in detoxification of toxic epoxides, was determined using human liver microsomes. Honokiol and β-amyrin acetate, isolated from Magnolia officinalis and Acer mandshuricum, respectively, displayed strong inhibition of sEH activity, with respective IC₅₀ values of 0.57 μM and 3.4 μM determined using the fluorescent substrate, and 1.7 μM and 6.1 μM determined using 14,15-epoxyeicosatrienoic acid. mEH activity was decreased to 49% or 61% of control activity by 25 μM honokiol or β-amyrin acetate, respectively. These results suggest that β-amyrin acetate and honokiol exhibit sEH inhibitory activity, although their sEH selectivity should be improved.     

Xenobiotic metabolizing enzyme activities in rat, mouse, monkey, and human testes.

The capacity of the testis to metabolize xenobiotics has been proposed to play a role in the susceptibility of different species to testicular toxicity. Since species differences in testicular xenobiotic metabolizing enzyme activities are not well documented, the primary objective of the present study was to compare enzyme activities in subcellular fractions prepared from rat, mouse, monkey, and human testes. In microsomal fractions, enzyme activities measured were pentoxyresorufin O-dealkylase (PROD), ethoxyresorufin O-dealkylase (EROD), and epoxide hydrolase (mEH). In cytosolic preparations, epoxide hydrolase (cEH) and glutathione S-transferase (cGST) activities were measured. PROD activity was not detectable in any of the species studied, while it was readily detected in liver microsomes used as a positive control. Although EROD activity was low, it was measurable in testicular microsomes from rat and mouse, but not monkey or human. No marked species differences in cEH activity were found. In contrast, mEH activity was low in the monkey, intermediate in the rat, and highest in the human and mouse. cGST activity was significantly lower in the two primate species compared with the rat and the mouse. The levels of activity of the xenobiotic metabolizing enzymes studied were generally more than an order of magnitude lower in the testis as compared to the liver. However, in rat and mouse, the levels of mEH and cGST activities in testis were relatively similar to hepatic levels. Overall, these data indicate that species differences in capacity to metabolize xenobiotics may play a role in differential sensitivity to testicular toxicants.     

The soluble epoxide hydrolase as a pharmaceutical target for hypertension

The soluble epoxide hydrolase appears to be a promising target for the development of antihypertensive therapies based on a previously unexplored mechanism of action. Epoxide hydrolases are enzymes that add water to three membered cyclic ethers known as epoxides. The soluble epoxide hydrolase in mammalian systems (sEH) is a member of the alpha/beta-hydrolase fold family of enzymes and it shows a high degree of selectivity for epoxides of fatty acids. The regioisomeric epoxides of arachidonic acid or epoxyeicosanoids (EETs) are particularly good substrates. These EETs appear to be major components of the endothelium-derived hyperpolarizing factors (EDHFs). As such, EETs cause vasodilation and reduce blood pressure. The EETs also are strongly anti-inflammatory and analgesic. By inhibiting sEH, the increase in circulating EETs leads to a reduction in blood pressure in a number of animal models. Potent transition state mimic inhibitors have been developed for the sEH. Some of these sEH inhibitors (sEHIs) show nanomolar to picomolar potency and good pharmacokinetic properties. Because of their unique mode of action they show promise in treating hypertension while reducing problems with end organ failure, vascular inflammation and diabetes. Indeed, the anti-inflammatory properties of the sEHI may make them particularly suitable for treating hypertension in patients with other concomitant metabolic syndromes. They are more potent on a molar basis than most nonsteroidal anti-inflammatory drugs (NSAIDs) in reducing PGE2 in inflammation models, they strongly synergize with NSAIDs, and appear to ameliorate apparently unfavorable eicosanoid profiles associated with some cyclo-oxygenase-2 inhibitors.     

1,2-Epoxycycloalkanes: substrates and inhibitors of microsomal and cytosolic epoxide hydrolases in mouse liver

Six different 1,2-epoxycycloalkanes, whose rings were constituted of 5 to 12 carbon atoms, were tested as possible inhibitors of epoxide-metabolizing enzymes and substrates for the microsomal and cytosolic epoxide hydrolases (mEH, cEH) in mouse liver. The geometric configurations and the relative steric hindrances of these epoxides were estimated from their ease of hydrolysis in acidic conditions to the corresponding diols, their abilities to react with nitrobenzylpyridine, and the chemical shifts of the groups associated with the oxirane rings measured by proton and 13C-NMR. The cyclopentene, -hexene, -heptene, -octene and -decene oxides adopted mainly a cis-configuration. By contrast, cyclododecene oxide presented a trans-configuration. Steric hindrance increased with the size of the ring and was particularly strong when cyclooctene, -decene and -dodecene oxides were considered. With the exception of cyclohexene oxide, all the compounds were weak inhibitors of EH and glutathione S-transferase (GST) activities. Cyclohexene oxide exhibited a selective inhibition of the mEH with an I50 of 4.0.10(-6) M. As the size of the ring increased, inhibitory potency was gradually lost. The cEH and the GST activities were less sensitive to the inhibitory effects of these epoxides (I50, 1 mM or above). A marked difference between the substrate selectivities of mEH and cEH for these epoxides was observed. The mEH hydrated all of the cyclic epoxides, although some of them at a very low rate; the best substrate was the cycloheptene oxide (2.3 nmol/min/mg protein). On the other hand, cyclodecene oxide was a substrate of cEH, but no diol formation was detected when cyclopentene, -hexene and -dodecene oxides were incubated with cytosolic enzyme.     

Soluble epoxide hydrolase: a novel target for the treatment of hypertension

Epoxide hydrolases are a group of enzymes that convert the epoxide group of chemical compounds to corresponding diols by the addition of water. Soluble epoxide hydrolase (sEH, formerly referred to as cytosolic epoxide hydrolase), which is widely distributed in mammalian tissues, is the primary enzyme responsible for the conversion of epoxyeicosatrienoic acids (EETs), the bioactive lipid mediators formed from arachidonic acid by cytochrome P450 epoxygenase, to their corresponding diols. EETs, but not their diols, are endogenous anti-hypertensive eicosanoids. Disruption of the sEH gene in male mice decreases blood pressure, and inhibition of sEH decreases blood pressure in several experimental hypertensive models. Potent selective sEH inhibitors have been developed, and these sEH inhibitors have potential to become a novel class of anti-hypertensive drug.     

Functional analysis of drug metabolizing enzymes using gene knockout animals

Several gene knockout mice have been widely used to analyze the role of drug-metabolizing enzymes in pharmacologic and physiologic responses. The metabolic shift of endogenous and exogenous compounds causes pharmacologic and physiologic alterations. Microsomal epoxide hydrolase (mEH)-null mice are less susceptible to the skin tumorigenesis, splenic immunotoxicity, and embryonic toxicity of 7,12-dimethylbenz[a] anthracene (DMBA). The production of DMBA-3,4-diol is detected in the target organs of wild-type mice, but not in those of mEH-null mice. Soluble epoxide hydrolase (sEH)-null mice exhibit markedly reduced rates of epoxyeicosatrienoic acid conversion to dihydroxyei-cosatrienoic acid in the liver and kidney. Furthermore, sEH-null male mice have a lower blood pressure phenotype compared with male wild-type mice, suggesting the importance of sEH in blood pressure regulation. Nuclear bile acid receptor, farnesoid X receptor (FXR)-null mice are distinguished from wild-type mice by elevated bile acid levels in the liver and serum. However, hepatic lithocholic acid (LCA) levels are lower in LCA-fed FXR-null female mice compared to those in wild-type female mice. Furthermore, FXR-null female mice are less susceptible to liver damage by LCA compared with female wild-type mice. Marked increases in hepattic LCA-sulfating activity and hepatic hydroxysteroid sulfotransferase and biliary sulfated bile acid levels are detected in FXR-null female mice, suggesting the protective role of hydroxysteroid sulfotransferase in LCA-induced liver damage. These and other studies indicate that mice null for drug-metabolizing enzymes and nuclear receptors are of great value in the study of the role of drug-metabolizing enzymes in pharmacologic and physiologic responses.     

Distribution and inducibility of cytosolic epoxide hydrolase in male Sprague-Dawley rats

Cytosolic epoxide hydrolase (cEH) activity has been determined in liver and various extrahepatic tissues of male Sprague-Dawley rats using trans-stilbene oxide (TSO) and trans-ethylstyrene oxide (TESO) as substrates. Large interindividual differences in the specific activity of cytosolic epoxide hydrolase in the liver from more than 80 individual rats were observed varying by a factor of 38. In a randomly selected group of five animals liver cEH varied by a factor of 3.9 and kidney cEH by a factor of 2.7, whereas liver microsomal epoxide hydrolase and lactate dehydrogenase showed only very low variations (1.4- and 1.1-fold, respectively). The individual relative activity of kidney cEH was related to that of the liver. Cytosolic epoxide hydrolase activity was present in all of six extrahepatic rat tissues investigated. Interestingly specific activities were very high in the heart and kidney (higher than in liver), followed by liver greater than brain greater than lung greater than testis greater than spleen. TSO and TESO hydrolases in subcellular fractions of rat liver were present at highest specific activities in the cytosolic and the heavy mitochondrial fraction. As indicated by the marker enzymes, catalase, urate oxidase and cytochrome oxidase, this organelle-bound epoxide hydrolase activity may be of peroxisomal and/or mitochondrial origin. In the microsomal fraction, TSO and TESO hydrolase activity is very low, whereas STO hydrolase activity is highest in this fraction and very low in cytosol. In kidney, subcellular distribution is similar to that observed in liver. None of the commonly used inducers of xenobiotic metabolizing enzymes caused significant changes in the specific activities of rat hepatic cEH (trans-stilbene oxide, alpha-pregnenolone carbonitrile, 3-methylcholanthrene, beta-naphthoflavone, isosafrole, butylated hydroxytoluene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dibenzo[a,h]anthracene, phenobarbitone). However, clofibrate, a hypolipidemic agent, very strongly induced rat liver cEH (about 5-fold), whereas microsomal epoxide hydrolase activity was not affected. Specific activity of kidney cEH was increased about 2-fold.     

Soluble epoxide hydrolase in rat inflammatory cells is indistinguishable from soluble epoxide hydrolase in rat liver.

Soluble epoxide hydrolase (sEH) is a ubiquitous mammalian enzyme for which liver and kidney are reported to have the highest activity. We have shown that the soluble epoxide hydrolase (sEH) activity present in rat neutrophils and macrophages is kinetically, immunologically, and physically indistinguishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.     

Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri. Baseline levels and optimization of assay conditions

Microsomal and cytosolic epoxide hydrolase (mEH and cEH respectively) and glutathione S-transferase (GST) activities were measured in the liver, kidney, and gills of rainbow trout. Assays were optimized for time, pH, and temperature, using trans-stilbene oxide (TSO) and cis-stilbene oxide (CSO) as substrates for cEH and mEH, respectively. Optimal pH values for mEH, cEH, and GST were similar to mammalian values (i.e. 8.5, 7.5, and 9). Temperature optima differed between tissues and cell fractions. Specific activity of cEH-TSO was 3-14 times greater than mEH-CSO for all three tissues, and 8-60 times greater on a tissue weight basis. Liver and, to a lesser extent, kidney mEH were active against benzo[a]pyrene 4,5-oxide, whereas gill mEH was not active against this substrate. Liver cytosolic GST was active against CSO and 1-chloro-2,4-dinitrobenzene (CDNB) but not TSO, whereas gill and kidney cytosolic GST were active only against CDNB. Liver and kidney microsomal GST were active against CDNB, but no activity was found in gill microsomes. The results are discussed in relation to possible endogenous substrates and uninduced xenobiotic metabolizing capacities of different trout tissues.     

环氧化物水解酶在高血压治疗中的作用及其机制研究进展

目的介绍环氧化物水解酶及其抑制物的理化性质及其在抗高血压治疗中的作用和机制。方法查阅相关文献,总结归纳环氧化物水解酶及其抑制物的理化性质和作用机制。结果可溶性环氧化物水解酶(sEH)广泛存在于哺乳动物组织中,是内源性抗高血压物质——二十碳脂酸(EET)的主要转换酶,破坏sEH基因或抑制sEH可引起血压下降。结论sEH抑制剂可能成为抗高血压的新药。     

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.     

Cardiovascular therapeutic aspects of soluble epoxide hydrolase inhibitors

Soluble epoxide hydrolase (sEH) is an enzyme responsible for the conversion of lipid epoxides to diols by the addition of water. Biological actions on the cardiovascular system that are attributed to epoxides include vasodilation, antiinflammatory actions and vascular smooth muscle cell antimigratory actions. Conversion of arachidonic acid epoxides to diols by sEH diminishes the beneficial cardiovascular properties of these epoxyeicosano-ids. Cardiovascular diseases in animal models and humans have been associated with decreased epoxygenase activity or increased sEH activity and these changes are responsible for the progression of the disease state. More recently, sEH gene polymorphisms in the human population have been associated with increased risk for cardiovascular diseases. Thus the biological actions of epoxyeicosanoids and the sEH enzyme are ideal therapeutic targets for cardiovascular diseases. The rapid development of 1,3-disubstituted urea based sEH inhibitors over the past five years has resulted in a number of studies demonstrating cardiovascular protection. sEH inhibitors have antihypertensive and antiinflammatory actions and have been demonstrated to decrease cerebral ischemic and renal injury in rat models of hypertension. These findings of beneficial actions in animal models of disease position the sEH enzyme as a promising therapeutic target for cardiovascular diseases.     

Inhibition of catalase and epoxide hydrolase by the renal cystogen 2-amino-4,5-diphenylthiazole and its metabolites

Subchronic feeding of 2-amino-4,5-diphenylthiazole (DPT) to rats results in the development of renal cysts and has been used as a model system to study polycystic kidney disease. Because previous studies revealed changes in renal enzymes following DPT administration, a possible direct effect of DPT and its phenolic metabolites on catalase and a related enzyme, epoxide hydrolase, was examined. Experiments with three in vitro systems (suspensions of rabbit renal tubules, rat kidney homogenates, and commercially obtained bovine liver catalase) revealed direct inhibition of catalase activity by the diphenolic metabolite (diOH- DPT: 2-amino-4,5di(4′-hydroxyphenyl)-thiazole), the known renal cystogen nordihydroquaiaretic acid (NDGA) 2-amino-4(4′- hydroxyphenyl),5-phenyl-thiazole (4OH-DPT), and the known catalase inhibitor 3-amino-1,2,4-triazole; DPT did not inhibit catalase activity. Following oral administration to rats of the DPT congeners, 4OH-DPT caused the greatest decrease in both renal catalase and cytosolic epoxide hydrolase activityes and the shortest time to onset of cystic lesions. In vitro, mouse liver cytosolic epoxide hydrolase activity was substantially inhibited by 4OH-DPT and dioH-DPT, and NDGA, but not by 2-amino-4-phenyl,5- (4′-hydroxyphenyl)-thiazole (5OH-DPT) or DPT itself. Microsomal epoxide hydrolase (mEH) activity was inhibited by 4OH-DPT, unaffected by DPT or dioH-DPT, and stimulated 2-fold by 5OH-DPT. Finally, mEH activity was substantially higher in samples of normal human kidney than in samples of kidney derived from a patient with autosomal recessive polycystic kidney disease; no differences were observed in cEH activity in these samples. Although the role of altered catalase and epoxide hydrolase activities in cystogenesis is unknown, DPT-induced cyst formation is associated with loss of these enzyme activities in kidney tissue. To our knowledge, this is the first report of an in vivo diminution of cytosolic epoxide hydrolase activity by xenobiotics.     

Studies on the developmental and adrenal regulation of cytosolic and microsomal epoxide hydrolase activities in rat liver.

The present study was undertaken to ascertain developmental profiles of microsomal epoxide hydrolase (mEH) and cytosolic epoxide hydrolase (cEH) enzyme activities in rat liver. During development, mEH activity reached an optimum by 6 weeks of age (63 nmol/min/mg protein). Activities decreased thereafter in both sexes although in adult male liver the activity was twice that measured in adult female liver. Thus, the importance of pituitary maturation was suggested from these findings. Since glucocorticoids have been implicated in the regulation of mEH gene expression the effect of adrenalectomy on mEH activity was investigated in adult male rat liver. The procedure increased mEH activity almost two-fold and the increase was reversed by dexamethasone, but not deoxycorticosterone, administration. With respect to hepatic cEH activity, the developmental profiles indicated that enzyme activity was greatest in rats at 1 week of age (12-15 nmol/min/mg protein) and very little activity was detected beyond 4 weeks of age (approximately 5 nmol/min/mg protein); sex differences in cEH activity were not apparent at any age. Thus, the pituitary appears to be important in the developmental induction of mEH but not cEH. Glucocorticoids appear to provide the major hormonal influence on mEH expression. Thus, the hypothalamus-pituitary-adrenal axis is involved in the regulation of mEH but the regulation of the cEH enzyme remains unclear.     

Characterization of DNA adducts derived from (+/- )-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4- tetrahydrodibenz[a,j]anthracene and (+/- )-7-methyl-trans-3,4-dihydroxy- anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene

Structural characterizations of the DNA adducts derived from reaction of the racemic bay region anti-diol epoxides of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene with calf thymus DNA are presented. Quantities of adducts necessary for spectroscopic characterization were obtained from reactions of the respective diol epoxides with individual deoxyribonucleotides. Both hydrocarbon diol epoxides showed similar adduct profiles upon reaction with calf thymus DNA in vitro which were composed mainly of three deoxyguanosine and four deoxyadenosine adducts. No significant modification of pyrimidine bases in DNA was detected with either of the diol epoxides. Approximately 3 times more deoxyguanosine than deoxyadenosine residues in the DNA were found to be modified by both diol epoxides. The DNA reactions showed very similar stereo- and enantioselectivities with both diol epoxides. The stereochemistries of addition of the purine bases to the diol epoxides were determined from analysis of the NMR spectra of individual adducts. The predominant adducts formed were products of trans addition of the exocyclic amino group of purines to the diol epoxides. The enantiomeric nature of the various adducts was determined from reaction of the individual deoxyribonucleotides with the pure (+)-anti-diol epoxide of dibenz[a,j]anthracene. The major deoxyguanosine and deoxyadenosine adducts from reactions with DNA were found to arise from the (+)-enantiomer of both hydrocarbon diol epoxides. The high reactivities of both diol epoxides (24-38%) with DNA in solution are consistent with the high tumor-initiating activity exhibited by the diol epoxide of dibenz[a,j]anthracene relative to the parent hydrocarbon.     

Haplotype structures of EPHX1 and their effects on the metabolism of carbamazepine-10,11-epoxide in Japanese epileptic patients

Objective Microsomal epoxide hydrolase (mEH) is an enzyme that detoxifies reactive epoxides and catalyzes the biotransformation of carbamazepine-10,11-epoxide (CBZ-epoxide) to carbamazepine-10,11-diol (CBZ-diol). Utilizing single nucleotide polymorphisms (SNPs) of the EPHX1 gene encoding mEH, we identified the haplotypes of EPHX1 blocks and investigated the association between the block haplotypes and CBZ-epoxide metabolism.Methods SNPs of EPHX1 were analyzed by means of polymerase chain reaction amplification and DNA sequencing using DNA extracted from the blood leukocytes of 96 Japanese epileptic patients, including 58 carbamazepine-administered patients. The plasma concentrations of CBZ and its four metabolites were determined using high-performance liquid chromatography.Results From sequencing all 9 exons and their surrounding introns, 29 SNPs were found in EPHX1. The SNPs were separated into three blocks on the basis of linkage disequilibrium, and the block haplotype combinations (diplotypes) were assigned. Using plasma CBZ-diol/CBZ-epoxide ratios (diol/epoxide ratios) indicative of the mEH activity, the effects of the diplotypes in each EPHX1 block were analyzed on CBZ-epoxide metabolism. In block 2, the diol/epoxide ratios increased significantly depending on the number of haplotype *2 bearing Y113H (P=0.0241). In block 3, the ratios decreased depending on the number of haplotype *2 bearing H139R (P=0.0351). Also, an increasing effect of a *1 subtype, *1c, was observed on the ratio.Conclusion These results show that some EPHX1 haplotypes are associated with altered CBZ-epoxide metabolism. This is the first report on the haplotype structures of EPHX1 and their potential in vivo effects.