Search

Cardiovascular and heart diseases are leading causes of morbidity and mortality. Coronary artery endothelial and vascular dysfunction, inflammation, and mitochondrial dysfunction contribute to progression of heart diseases such as arrhythmias, congestive heart failure, and heart attacks. Classes of fatty acid epoxylipids and their enzymatic regulation by soluble epoxide hydrolase (sEH) have been implicated in coronary artery dysfunction, inflammation, and mitochondrial dysfunction in heart diseases. Likewise, genetic and pharmacological manipulations of epoxylipids have been demonstrated to have therapeutic benefits for heart diseases. Increasing epoxylipids reduce cardiac hypertrophy and fibrosis and improve cardiac function. Beneficial actions for epoxylipids have been demonstrated in cardiac ischemia reperfusion injury, electrical conductance abnormalities and arrhythmias, and ventricular tachycardia. This review discusses past and recent findings on the contribution of epoxylipids in heart diseases and the potential for their manipulation to treat heart attacks, arrhythmias, ventricular tachycardia, and heart failure., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are the most prevalent metabolic liver disorders and a serious global health burden. NAFLD/NASH pathogenesis and progression are highly multi-factorial and likely demand a combination of multiple mechanisms to provide a more effective treatment. We have developed a dual farnesoid X receptor agonist (FXRA)/soluble epoxide hydrolase inhibitor (sEHi) to simultaneously address two validated and complementary modes of action in NASH treatment. Here we report the in vivo profiling for this FXRA/sEHi in toxin- and diet-induced rodent NASH models. In streptozotocin-induced NASH as a proof-of-concept study, the experimental FXRA/sEHi drug robustly prevented hepatic steatosis and fibrosis, and improved lipid homeostasis as well as biochemical markers of liver health. In methionine-choline-deficient high-fat diet-induced NASH, FXRA/sEHi treatment reduced hepatic steatosis and fibrosis to levels similar to healthy animals and demonstrated anti-inflammatory activity confirming that dual FXRA/sEHi modulation produces a triad of complementary anti-NASH effects. Our results validate dual FXRA/sEHi modulation as an effective therapeutic strategy to treat NASH and advocates for a combinational drug therapeutic approach for multifactorial liver diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

N,N'-disubstituted urea-based soluble epoxide hydrolase (sEH) inhibitors are promising therapeutics for hypertension, inflammation, and pain in multiple animal models. The drug absorption and pharmacological efficacy of these inhibitors have been reported extensively. However, the drug metabolism of these inhibitors is not well described. Here we reported the metabolic profile and associated biochemical studies of an N-adamantyl urea-based sEH inhibitor 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) in vitro and in vivo. The metabolites of AEPU were identified by interpretation of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or NMR. In vitro, AEPU had three major positions for phase I metabolism including oxidations on the adamantyl moiety, urea nitrogen atoms, and cleavage of the polyethylene glycol chain. In a rodent model, the metabolites from the hydroxylation on the adamantyl group and nitrogen atom were existed in blood while the metabolites from cleavage of polyethylene glycol chain were not found in urine. The major metabolite found in rodent urine was 3-(3-adamantyl-ureido)-propanoic acid, a presumably from cleavage and oxidation of the polyethylene glycol moiety. All the metabolites found were active but less potent than AEPU at inhibiting human sEH. Furthermore, cytochrome P450 (CYP) 3A4 was found to be a major enzyme mediating AEPU metabolism. In conclusion, the metabolism of AEPU resulted from oxidation by CYP could be shared with other N-adamantyl-urea-based compounds. These findings suggest possible therapeutic roles for AEPU and new strategies for drug design in this series of possible drugs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects., (Published by Elsevier Inc.)

The soluble epoxide hydrolase (sEH) is involved in the metabolism of arachidonic, linoleic, and other fatty acid epoxides, endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas, carbamates, and amides are new potent and stable inhibitors of sEH. However, the poor solubility of the lead compounds limits their use. Inhibitor structure-activity relationships were investigated to better define the structural requirements for inhibition and to identify points in the molecular topography that could accept polar groups without diminishing inhibition potency. Results indicate that lipophilicity is an important factor controlling inhibitor potency. Polar groups could be incorporated into one of the alkyl groups without loss of activity if they were placed at a sufficient distance from the urea function. The resulting compounds had a 2-fold higher water solubility. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties.

Previous studies in this laboratory have shown that oltipraz (Olt), a chemopreventive agent, enhances radiation(Rad)-inducible glutathione S-transferase (GST) and microsomal epoxide hydrolase (mEH) expression in the liver. The present study was designed to investigate the in vivo radioprotective effect of Olt in ICR mice exposed to a lethal dose of Rad. The 30-day survival rate of mice irradiated at the dose of 8 Gy was substantially increased to 91% by Olt pretreatment (100 mg/kg/day for 2 days), compared with 48% in animals irradiated alone. Light microscopic examinations revealed that exposure of mice to 8 Gy of gamma-ray Rad resulted in hepatocyte degeneration in the surviving animals from Day 1 through Day 22 after irradiation with certain degrees of necrosis observed at early times, whereas Olt treatment provided protection of the liver against irradiation with no hepatic necrosis noted. Mice irradiated at the dose of 8 Gy exhibited time-related decreases in the white blood cell (WBC), red blood cell (RBC), and platelet counts with maximal reduction noted at Day 10. Animals irradiated with Olt treatment showed no difference in peripheral blood cell counts or in the ratio of myeloid to erythroid bone marrow cells, compared with those irradiated alone. Northern RNA blot analysis showed that treatment of mice with Olt at the dose of 100 mg/kg in combination with 8 Gy irradiation resulted in 12-fold increases in hepatic mEH and mGSTA3 mRNA levels at 24-hr post-treatment, whereas mGSTP1 mRNA levels were not altered. The mRNA levels for mEH and mGSTA3 were elevated after exposure of animals to both Olt and 8 Gy-gamma ray to a greater extent than after irradiation alone. The enhanced survival rate (91%) in 8 Gy-irradiated animals after treatment with Olt (100 mg/kg/day for 2 days) was completely reversed by concomitant pretreatment with dexamethasone (Dexa) (0.1 and 1 mg/kg/day for 2 days), a known inhibitor of mEH and GST expression, resulting in a 42% and 28% survival rate, respectively. Mice irradiated after dexamethasone treatment at a dose of 1 mg/kg showed a reduced mean survival time compared with those treated with 0.1 mg/kg of dexamethasone (9 vs 14 days). The current study demonstrates that Olt is effective in increasing the survival rate of mice against ionizing Rad and that protective effects of Olt associated with enhanced expression of mEH and GST genes may represent its radioprotective efficacy.

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. To investigate the role of LTB4 in dermatitis, we used S-(4-dimethylaminobenzyl)-N-[(2S)-3-mercapto-2-methylpropionyl]-L- cysteine (SA6541), a potent leukotriene A4 (LTA4) hydrolase inhibitor. SA6541 inhibited LTB4 production with an IC50 value of 270 nM in vitro. 5-Hydroperoxyeicosatetraenoic acid (5-HPETE) or AA injection induced LTB4 production and neutrophil influx in mouse ear. SA6541 inhibited 5-HPETE- and AA-induced LTB4 production and neutrophil influx in mouse ear when administered orally at a dose of 50 mg/kg. SA6541 also inhibited 5-HPETE-induced prostaglandin E2 (PGE2) production, probably by an indirect effect through the inhibition of LTB4 production. These results suggest that LTB4 may be important in the pathogenesis of dermatitides such as psoriasis.

Idiosyncratic hypersensitivity reactions with carbamazepine have been postulated to be due to a deficiency of microsomal epoxide hydrolase (HYL1), although this is based on indirect evidence. Using 3H-cis stilbene oxide (0.5 Ci/mmol) as a substrate, we have developed a radiometric HPLC assay sensitive enough to measure the kinetic parameters of HYL1 in lymphocytes. The intra-assay coefficient of variation was 8%. Enzyme activity has been measured in lymphocytes from six carbamazepine hypersensitive patients, six patients on carbamazepine without any adverse effects, and twelve drug-naive healthy volunteers. No significant difference was observed in three kinetic parameters of the enzyme among these three groups. The values for Km, Vmax, and intrinsic clearance ranged from 6.1-89.9 microM, 3.0-23.2 pmoles diol formed/min/mg protein, and 0.147-0.493 microliter/min/mg protein. There was no difference in enzyme activity between patients currently on carbamazepine and healthy volunteers, indicating a lack of induction of lymphocyte HYL1 by carbamazepine. Co-incubation of lymphocytes with 1,1,1-trichloropropene oxide, an inhibitor of hepatic HYL1, resulted in an 82% inhibition of activity, similar to that observed with the hepatic enzyme. The healthy volunteers were genotyped as being either GSTM1 positive (n = 6) or GSTM1 negative (n = 6). This did not affect the kinetic parameters of lymphocyte microsomal epoxide hydrolase. Our results suggest that there is normal HYL1 activity in lymphocytes of hypersensitive patients using cis-stilbene oxide as a substrate.

Human neutrophil leukotriene A4 (LTA4) hydrolase is a zinc-containing metalloproteinase with aminopeptidase activity and can be inhibited by some metalloproteinase inhibitors. Human airway epithelial cells also contain an LTA4 hydrolase enzyme that has some novel properties, suggesting that this enzyme may be functionally and structurally unique. Thus, we questioned whether the epithelial cells were studied either intact or disrupted. Of the metalloproteinase inhibitors examined, only captopril, bestatin, and fosinoprilat had appreciable inhibitory activity for LTA4 hydrolase in disrupted epithelial cells. Concentration-inhibition curves to captopril, bestatin, and fosinoprilat revealed IC50 values of 430 microM, 7 microM, and 1 mM, respectively, for disrupted-cell LTA4 hydrolase activity. In contrast to its effects on neutrophils, 1,10-O-phenanthroline had no significant effect on disrupted epithelial cell hydrolase activity and had only minimal effects when this activity was partially purified (179-fold). LTA4 hydrolase concentration-inhibition curves examined in intact cells with captopril, bestatin, and 1,10-O-phenanthroline revealed IC50 values of 63, 70, and 920 microM, respectively. Aminopeptidase activity in disrupted epithelial cells was inhibited by amastatin, bestatin, and 1,10-O-phenanthroline (IC50 values of 500 nM, 1 microM, and 17 microM, respectively), but not by captopril at the highest concentration tested, 10 mM. These findings are in contrast to prior studies in neutrophils. When neutrophils were stimulated with A23187 after treatment with captopril, transcellular synthesis of LTB4 was inhibited more effectively than direct synthesis of leukotriene B4 (LTB4) (43.8 +/- 2.5 vs 18.5 +/- 4.7%; N = 8, P < 0.02). We conclude that LTA4 hydrolase activity of human airway epithelial cells is inhibited by some metalloproteinase inhibitors, but that the profile of inhibition is distinct from that for the neutrophil enzyme. These data provide additional information that LTA4 hydrolase in the epithelial cell is a novel enzyme, distinct from that found in the neutrophil.

We investigated the effects of bestatin, a prototype leukotriene A4 (LTA4) hydrolase inhibitor, on leukotriene (LT) formation and pulmonary artery perfusion pressure (Ppa) in isolated, perfused rat lungs. In lung parenchymal strips stimulated with a 10 microM concentration of the Ca2+ ionophore A23187, bestatin inhibited LTB4 formation with an IC50 = 10.4 +/- 30 microM (mean +/- SD, N = 4). It did not alter cysteinyl LT formation, confirming that it inhibited LTA4 hydrolase selectively, without inhibiting phospholipase, 5-lipoxygenase, or LTC4 synthase. In isolated, perfused lungs stimulated with 10 microM A23187, 300 microM bestatin inhibited LTB4 release by 72.2 +/- 10.6% (mean +/- SEM, N = 6, P < 0.01) but had no significant effect on LTE4 formation (P > 0.5). In these perfused lungs, bestatin did not alter the change in Ppa following stimulation with A23187. This effect is consistent with the insubstantial re-direction of LTA4 toward formation of vasospastic cysteinyl LTs. Separate experiments used lungs from rats treated with lipopolysaccharide endotoxin in vivo, prior to isolation, perfusion, and stimulation with 5 microM formyl-methionyl-leucyl-phenylalanine, in vitro. In these inflamed lungs, 750 microM bestatin inhibited LTB4 formation (P < 0.05) and increased LTE4 formation (P < 0.05), compatible with selective inhibited LTB4 hydrolase. The re-direction of LTA4 metabolism toward formation of cysteinyl LTs by inflamed, perfused lungs did not cause an increase in P(pa).

Valproic acid (VPA) and its analogues valpromide (VPM), valproyl-Coenzyme A (VP-CoA) and valproyl-ethylester (VPE) were examined as potential inhibitors of microsomal epoxide hydrolase (mEHb) using styrene-7,8-oxide (STO) and benzo(a)pyrene-4,5-oxide (BPO) as enzyme substrates. The effect of each potential inhibitor was examined using mEHb from rat liver, human livers (from a child, woman and man) and from human placenta. Of the compounds tested, only VPM (2 mM) expressed significant inhibition of mEHb activity with a maximum inhibition of 49%, 48%, 35% and 33% for liver microsomes from the child, woman, man and rat, respectively, using STO (2 mM) as substrate. Human placenta mEHb was inhibited 59% under the same conditions. The inhibition was found to be competitive, with closely related KI values of 0.11, 0.16, 0.28, 0.27 and 0.31 mM for mEHb obtained from rat liver, human placenta, child, female and male liver, respectively. VPA demonstrated only a slight inhibition (maximum 16%) of mEHb at high concentrations (10 mM), and VP-CoA was found to activate STO hydrolysis slightly at concentrations between 1 and 5 mM. VPE caused a moderate concentration-dependent activation of mEHb in all microsomal preparations examined. The inhibitory or activating properties of each compound were independent of the substrate and influenced slightly by the pH used in the incubation medium. The lack of inhibition of mEHb by VPA and its analogues other than VPM shows that neither masking of the carboxyl function of VPA nor the introduction of higher lipophilicity are sufficient to account for the inhibitory properties of VPM for mEHb. A molecular mechanism for the inhibition of mEHb by VPM is discussed.

A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae. The heterologous enzyme was located mainly in the microsomal fraction of yeast cells. Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity. The hmEH catalysed exclusively the formation of carbamazepine-10,11-transdihydrodiol, since no carbamazepine-10,11-cisdihydrodiol was detected. Inhibition studies using these microsomes revealed unequivocally hmEH as the target for inhibition by the antiepileptic drug valpromide. A Ki value of 27 microM was determined for the inhibitor valpromide with styrene oxide as substrate. For carbamazepine epoxide, a Ki value of 8.6 microM was obtained, which is well in line with data published for hmEH determined with human liver microsomes. Our results demonstrate the potential of heterologous gene expression in S. cerevisiae and its application to the in vitro study of pharmacological and toxicological problems.

The inhibition of murine cytosolic epoxide hydrolase has been studied with both racemic and enantiomerically pure trans-3-phenylglycidols. These compounds are the first enantioselective, slow binding inhibitors of cytosolic epoxide hydrolase. The (2S,3S)-3-phenylglycidol enantiomer was always a better inhibitor than the (2R,3R)-enantiomer. When the I50 values of (2S,3S)- and (2R,3R)-3-(4-nitrophenyl)glycidol were compared, the (2S,3S)-enantiomer was at least a 750-fold better inhibitor (I50 = 1.6 microM) than the (2R,3R)-enantiomer (I50 = 1200 microM), and it was the most potent inhibitor tested in the 3-phenylglycidol series. If the hydroxyl group of the glycidol was masked or converted to another functionality, the potency of the inhibitor decreased and the (2S,3S)-enantiomer was not necessarily the better inhibitor. In addition, trans-3-phenylglycidols demonstrated slow binding inhibition of cytosolic epoxide hydrolase. Inhibitors without a hydroxyl group, or with a blocked hydroxyl group, were not slow binding inhibitors. These results suggested that the hydroxyl group was important in both enantioselectivity and time dependence of inhibition of cytosolic epoxide hydrolase by trans-3-phenylglycidols. The hydration pattern of (2S,3S)- and (2R,3R)-2,3-epoxy-3-(4- nitrophenyl)glycidol by cytosolic epoxide hydrolase also differed. When incorporation of [18O] from water catalyzed by cytosolic epoxide hydrolase was measured, the (2S,3S)-enantiomer gave 12% incorporation into the benzylic carbon and the (2R,3R)-enantiometer gave 40% incorporation into the benzylic carbon. Finally, trans-3-phenylglycidols were found to be poor inhibitors of microsomal epoxide hydrolase.

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.

The inhibition in vitro and induction in vivo of microsomal trans-stilbene oxide hydrolase have been studied. This microsomal epoxide hydrolase activity is distinguishable from the previously well-defined microsomal arene oxide hydrolase by a number of catalytic criteria. Two substituted chalcone oxides, 4-phenylchalcone oxide and 4'-phenylchalcone oxide, are potent inhibitors of microsomal trans-stilbene oxide hydrolase, but have no apparent activity against benzo[a]pyrene 4,5-oxide hydrolase. Conversely, compounds that are potent inhibitors of benzo[a]pyrene 4,5-oxide hydrolase, including styrene oxide, cyclohexene oxide, and trichloropropene oxide, inhibit microsomal trans-stilbene oxide hydrolase only at very high (millimolar) concentrations. The chalcone oxides inhibit microsomal trans-stilbene oxide hydrolase noncompetitively, and have micromolar or nanomolar affinity constants for the enzyme. Attempts were made to induce microsomal trans-stilbene oxide hydrolase in vivo. Compounds that induced microsomal benzo[a]pyrene 4,5-oxide hydrolase levels in mice did not simultaneously induce trans-stilbene oxide hydrolase levels. Clofibrate was an exception; it induced levels of both enzymes to a small but statistically significant degree. The two microsomal hydrolase activities have, therefore, very different catalytic sites and appear to be under separate genetic control. 4-Phenylchalcone oxide and 4'-phenylchalcone oxide are selective inhibitors of microsomal trans-stilbene oxide hydrolase and may prove to be very useful in assessing the involvement of this enzyme in the metabolism of endogenous or xenobiotic epoxides.

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.

The initial rates of hydration of sixteen epoxides in the presence of cytosolic and microsomal fractions of mouse liver were determined. 1,2-Disubstituted trans-epoxides were found to be excellent, selective substrates for the cytosolic epoxide hydrolase, while 1,2-cis-epoxides were poorly hydrated when one or more substituents was a phenyl moiety. Epoxides of cyclic systems including benzo[alpha]pyrene 4,5-oxide, and two cyclodiene analogs were hydrated almost exclusively by the microsomal epoxide hydrolase while monosubstituted epoxides were hydrated by both systems. Some epoxides which were mediocre substrates proved to be reasonable inhibitors of the cytosolic epoxide hydrolase, indicating that the structural requirements for substrate binding and turnover are different. Some reagents known to interact with sulfhydryl groups, including styrene oxide, proved to be good inhibitors. This work facilitates the design of radiochemical and spectrophotometric assays for both major forms of epoxide hydrolase as well as prediction of potential intrinsic substrates. Also such data may be meaningful in assessing the risk involved in human exposure to epoxidized xenobiotics.

The cytotoxic properties of quinone drugs such as menadione and adriamycin are thought to be mediated through one-electron reduction to semiquinone free radicals. Redox cycling of the semiquinones results in the generation of reactive oxygen species and in oxidative damage. In this study the toxicity of mitozantrone, a novel quinone anticancer drug, was compared with that of menadione in human Hep G2 hepatoma cells. Mitozantrone toxicity in these cells was not mediated by the one-electron reduction pathway. In support of this, inhibition of the enzymes glutathione reductase and catalase, responsible for protecting the cells from oxidative damage, did not affect the response of the Hep G2 cells to mitozantrone, whereas it exacerbated menadione toxicity. In addition, the toxicity of menadione was preceded by depletion of reduced glutathione which was probably due to oxidation of the glutathione. Mitozantrone did not cause glutathione depletion prior to cell death. DT-diaphorase activity and intracellular glutathione were found to protect the cells from the toxicity of both quinones. Inhibition of epoxide hydrolase potentiated mitozantrone toxicity but did not affect that of menadione. Our experiments indicate that mitozantrone toxicity may involve activation to an epoxide intermediate. Both quinone drugs inhibited cytochrome P-450-dependent mixed-function oxidase activity, although menadione was more potent in this respect.

A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.