Hepatic Microsomal Epoxide Hydrolase Research Articles

A physiologically based toxicokinetic model for inhaled ethylene and ethylene oxide in mouse, rat, and human

Ethylene (ET) is the largest volume organic chemical. Mammals metabolize the olefin to ethylene oxide (EO), another important industrial chemical. The epoxide alkylates macromolecules and has mutagenic and carcinogenic properties. In order to estimate the EO burden in mice, rats, and humans resulting from inhalation exposure to gaseous ET or EO, a physiological toxicokinetic model was developed. It consists of the compartments lung, richly perfused tissues, kidneys, muscle, fat, arterial blood, venous blood, and liver containing the sub-compartment endoplasmic reticulum. Modeled ET metabolism is mediated by hepatic cytochrome P450 2E1, EO metabolism by hepatic microsomal epoxide hydrolase or cytosolic glutathione S-transferase in various tissues. EO is also spontaneously hydrolyzed or conjugated with glutathione. The model was validated on experimental data collected in mice, rats, and humans. Modeled were uptake by inhalation, wash-in–wash-out effect in the upper respiratory airways, distribution into tissues and organs, elimination via exhalation and metabolism, and formation of 2-hydroxyethyl adducts with hemoglobin and DNA. Simulated concentration-time courses of ET or EO in inhaled (gas uptake studies) or exhaled air, and of EO in blood during exposures to ET or EO agreed excellently with measured data. Predicted levels of adducts with DNA and hemoglobin, induced by ET or EO, agreed with reported levels. Exposures to 10000 ppm ET were predicted to induce the same adduct levels as EO exposures to 3.95 (mice), 5.67 (rats), or 0.313 ppm (humans). The model is concluded to be applicable for assessing health risks from inhalation exposure to ET or EO.

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep −/− mice

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2β-, 6β-, and 15β-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Induction of epoxide hydrolase and glucuronosyl transferase by isothiocyanates and intact glucosinolates in precision-cut rat liver slices: importance of side-chain substituent and chirality

The potential of three isothiocyanates, namely R,S-sulforaphane, erucin and phenethyl isothiocyanate, of two naturally occurring glucosinolates, namely glucoerucin and glucoraphanin, and of the enantiomers of sulforaphane to modulate glucuronosyl transferase and epoxide hydrolase, two major carcinogen-metabolising enzyme systems, was investigated in precision-cut rat liver slices. Following exposure of the slices to the isothiocyanates (0-25μM), erucin and phenethyl isothiocyanate, but not R,S-sulforaphane, elevated glucuronosyl transferase and epoxide hydrolase activities and expression, determined immunologically. Of the two enantiomers of sulforaphane, the R-enantiomer enhanced, whereas the S-enantiomer impaired, glucuronosyl transferase activity and only the former increased protein expression; furthermore, R-sulforaphane was more effective than the S-enantiomer in up-regulating microsomal epoxide hydrolase. When precision-cut rat liver slices were exposed to the same concentrations of glucoerucin and glucoraphanin, both glucosinolates caused a marked increase in the activity and expression of the microsomal epoxide hydrolase but had no effect on glucuronosyl transferase activity. It may be inferred that the ability of isothiocyanates to enhance hepatic microsomal epoxide hydrolase and glucuronosyl transferase activities is dependent on the nature of the side chain. Moreover, in the case of sulforaphane, the naturally occurring R-enantiomer increased both activities, whereas, in contrast, activities were impaired in the case of the S-enantiomer. Finally, intact glucosinolates are potent inducers of epoxide hydrolase and can thus contribute directly to the chemopreventive potential associated with cruciferous vegetable consumption.

Regulation of ATP‐binding cassette (Abc) transporters by organoselenium compounds

Selenium‐containing compounds have demonstrated chemopreventive activity in a variety of chemically‐induced tumor models, but a unifying mechanism for this effect remains elusive. Altered disposition of chemicals and toxic metabolites may be contributing factors. Following dietary ingestion of selenocysteine pro‐drugs and selenocystine (15 ppm Se) for 1 month, mouse hepatic glutathione transferase, quinone oxidoreductase and microsomal epoxide hydrolase activities were elevated by 2‐butyl‐ and 2‐oxoselenazolidine‐4‐carboxylic acids (SCAs) and selenocystine, but not by 2‐cyclohexyl‐, 2‐phenyl‐, and 2‐(2′‐hydroxyphenyl)‐ SCAs. Expression of multidrug resistance‐associated proteins and other Abc transporters were evaluated by qPCR. Of 10 transporters examined, none responded in a pattern similar to the activities of the enzymes capable of being regulated via the Nrf2/ARE mechanism. Abcc3 (Mrp3) was increased only by 2‐butylSCA and no transporters were induced by 2‐oxoSCA or selenocystine. 2‐(2′‐Hydroxyphenyl)SCA was the only compound that induced expression of Abcc2 (Mrp2) and Abcb6. All the selenocompounds reduced the expression of Abcb11. Expression of Abcc1, Abcc4, Abcc5, Abcc6, and Abcg2 and Abca3, was not altered by any compound. Selenocompound regulation of Abc transporter expression appears to be compound dependent and unrelated to the Nrf2/ARE pathway.Support: GM 058913

Characterization and profiling of hepatic cytochromes P450 and phase II xenobiotic-metabolizing enzymes in beluga whales ( Delphinapterus leucas) from the St. Lawrence River Estuary and the Canadian Arctic

Cytochromes P450 (CYP, phase I) and conjugating (phase II) enzymes can be induced by and influence the toxicokinetics (metabolism) and toxicity of xenobiotic contaminants in exposed organisms. Beluga whale ( Delphinapterus leucas) from the endangered St. Lawrence (SL) River Estuary population exhibit deleterious health effects and various severe pathologies that have been associated with contaminant exposure. In contrast, such effects (e.g. reproductive and immunological impairment) are generally less frequent in less exposed populations in the Canadian Arctic (CA). In the present study, opportunistic sampling resulted in the collection immediately after death of liver tissue from a single female neonate SL beluga (SL6) and male and female CA beluga ( n=10) from the Arviat region of western Hudson Bay, in addition to sampling of stranded carcasses of male and female SL beluga ( n=5) at least 12 h postmortem. We immunologically characterized cross-reactive proteins of hepatic microsomal CYP1A, CYP2B, CYP3A, CYP2E, epoxide hydrolase (EH) and uridine diphosphoglucuronosyl transferase (UDPGT) isozymes. Cross-reactive proteins were found in all SL and CA beluga using anti-rat CYP1A1, anti-rainbow trout CYP3A, anti-human CYP2E1, anti-rabbit EH and anti-human UDPGT1A1 polyclonal antibodies (Abs), whereas faintly cross-reactive CYP2B proteins were only found in SL6 and the CA samples using an anti-rabbit CYP2B1 Ab. In corresponding catalytic activity assessments, only SL6 and all CA beluga microsomal samples exhibited CYP1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activity (51–260 pmol/mg/min), CYP3A-mediated activity (113–899 pmol/mg/min) based on the formation of 6β-hydroxytestosterone using a testosterone hydroxylase assay, and UDPGT activity (830–4956 pmol/mg/min) based on 1-naphthylglucuronide formation. The marginal cross-reactivity with the anti-CYP2B1 Ab and lack of catalytically measurable hydroxytestosterone isomers associated with CYP2B-type activity in all the SL and CA animals is suggestive of low CYP2B-type enzyme expression in beluga. The absence of measurable total P450 enzyme levels and catalytic activities in samples from the stranded SL belugas suggested catalytically inactive enzymes as a consequence of tissue degradation related due to the time delay of sample collection after death. However, all SL and CA animals demonstrated similar, immunologically cross-reactive phase I and II hepatic enzyme profiles, which is suggestive of the importance of metabolism in the toxicokinetics and fate of xenobiotics in animals from both populations.

Xenobiotic conjugation systems in deer compared with cattle and rat

The ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzo[ a]pyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1-chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species.

The Ephx1(d) allele encoding an Arg338Cys substitution is associated with heat lability.

Heat lability of the mouse hepatic microsomal epoxide hydrolase 1 enzyme-specific activity (EC 3.3.2.3) is greater for the A/J than the C57BL/6J strain. Analysis of the microsomal epoxide hydrolase 1 cDNA coding sequences shows the C57BL/6J and A/J strains to differ in a single base, a C to T transition at position 1012 from the ATG. This change would predict a substitution of an Arg for a Cys at codon 338. Lyman et al. (J. Biol. Chem 255:8650, 1980) studied 26 inbred mouse strains and assigned each strain to one of two groups based upon functional criteria that included heat lability and pH optima for microsomal epoxide hydrolase 1. The heat-labile strains including A/J were denoted with the Ephx1(d) allele, whereas C57BL/6J and other members of the heat-stable strains were denoted with the Ephx1(b) allele. We examined those same inbred mouse strains and found complete concordance between the assignment of microsomal epoxide hydrolase 1 allele superscript "b" or "d" and the wild-type and C1012T polymorphism respectively (Fisher's Exact Test, two-sided p < 0.0001). These data suggest that mouse hepatic microsomal epoxide hydrolase 1 heat lability is associated with the presence of a Cys at residue 338. Genomic samples from the available AXB and BXA recombinant inbred strains were allelotyped for the SNP identified in the Ephx1 gene that distinguishes the A/J and C57BL/6J parental strains and used to map Ephx1 to Chromosome (Chr) 1 at approximately 98.5cM (LOD = 10.0).

Enhanced expression of rat hepatic microsomal epoxide hydrolase by methylthiazole in conjunction with liver injury

Microsomal epoxide hydrolase (mEH) is inducible by a number of xenobiotics. Induction of mEH by certain chemopreventive agents may implicate the protective effect. In contrast, many of carcinogenic agents also induce the enzyme. The hepatotoxicity and mEH expression by methylthiazoles, which are incorporated as functional groups in a number of therapeutic agents, were assessed in the rat liver to study the structural basis for the enzyme induction and the correlative enzyme expression with hepatotoxicity. Among the methylthiazoles examined, 4-methylthiazole (MT) at the daily dose of 1.17 mmol/kg body weight caused hepatic necrosis and degeneration after 1–3 consecutive daily treatment(s), whereas 4,5-dimethylthiazole (DT) and 2,4,5-trimethylthiazole (TT) elicited no toxicity. Treatment of rats with MT at the daily dose of 1.17 mmol/kg increased the mEH mRNA by 17- and 7-fold at day 1 and day 3, respectively, relative to control. Whereas DT caused 5- and 2-fold increases in mEH mRNA at day 1 and day 3, respectively, TT minimally affected mEH expression. The mRNA increase was consistent with the protein induction. Hence, the methylthiazole causing hepatotoxicity was more active in inducing the enzyme. Whereas treatment with MT at the dose of 0.35 mmol/kg caused no hepatotoxicity, MT caused hepatic necrosis in starving rats. Northern blot analysis showed that the mEH mRNA level was increased to a greater extent by MT in starving rats than in control animals. Conversely, treatment of starving rats with either cysteine or methionine prior to MT prevented the hepatic necrosis. Elevation of the mEH mRNA by MT in starving animals was also inhibited by either cysteine or methionine pretreatment. These results demonstrated that the methylthiazole which caused hepatotoxicity also up-regulated mEH expression, whereas other methylthiazoles showing no toxicity minimally increased the gene expression. The observation that the extent of mEH expression by MT was highly associated with that of liver injury raised the notion that mEH expression by xenobiotics may not necessarily represent the beneficial and protective effects.

Induction of hepatic xenobiotic metabolizing enzymes in female Fischer-344 rats following repeated inhalation exposure to decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D5-exposed animals showed significant recovery. Exposure to D5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2-fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6beta-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D5 vapors is similar to that reported for phenobarbital, and therefore D5 may be described as a weak "phenobarbital-like" inducer.

Thioureas differentially induce rat hepatic microsomal epoxide hydrolase and rGSTA2 irrespective of their oxygen radical scavenging effect: effects on toxicant-induced liver injury

Thioureas have been employed as potent hydroxyl radical scavengers and also inhibit production of oxygen free radicals. The in vitro oxygen radical scavenging effect by N, N′-substituted thioureas including dimethylthiourea (DMT), diethylthiourea (DET), tetramethylthiourea (TMT) and diphenylthiourea (DPT) was assessed by the conversion of φx-174 DNA from supercoiled DNA to the open circular form or to fragmented DNA. Addition of the N, N′-substituted thioureas to the incubation mixture significantly prevented a single strand breakage of φx-174 DNA induced by autooxidation of benzenetriol. These thioureas were also effective in preventing degradation of φx-174 DNA induced by autooxidation of benzenetriol in the presence of ferrous iron. In view of the in vitro radical scavenging effect by the thioureas and the role of reactive oxygen species in the induction of phase II detoxifying enzymes, expression of microsomal epoxide hydrolase (mEH) and rGSTA2 in response to these agents was investigated in the rat liver. Rats treated with each of the alkylthioureas exhibited marked increases of mEH and rGSTA2 mRNA levels with TMT being the most effective. DPT an arylthiourea, however, was minimally active in increasing the mRNAs. Time-course studies revealed that DMT, DET and TMT increased the mRNA levels to the greatest extent at 24 h after a single dose of treatment. The levels of mEH and rGSTA2 mRNA were elevated in a dose-dependent manner by the alkylthioureas. Immunoblot analysis showed that the alkylthioureas induced mEH and rGSTA2 proteins in the liver (0.6 mmol/kg per day, 3 days), which was consistent with the increases in the mRNA levels. DMT, DET or TMT enhanced CCl 4-induced liver toxicity, as monitored by plasma aminotransferase activity, although each of the agents alone caused only slight increase in the alanine aminotransferase activity. In contrast to the effects of the alkylthioureas, DPT protected the liver against the toxicant-induced injury. All of the thioureas prevented decreases in the hepatic glutathione level by CCl 4. Expression of cytochrome P450 2E1 and P450 2B1/2, which are implicated with metabolic activation of CCl 4, was assessed after treatment with the thioureas. P450 2E1 and P450 2B1/2 were differentially induced by the alkylthioureas with the expression of P450 2E1 being inversely related with that of P450 2B1/2. These results showed that N, N′-substituted alkylthioureas were capable of inducing mEH and rGSTA2 in the liver with elevation of the mRNAs, that induction of mEH and rGSTA2 by these alkylthioureas might be mediated by production of the reactive oxygens derived from metabolic activation of the agents irrespective of their radical scavenging effect and that the agents rather enhanced toxicant-induced liver injury with the induction of P450 2E1 or P450 2B1/2.

Lipopolysaccharide inhibition of rat hepatic microsomal epoxide hydrolase and glutathione S-transferase gene expression irrespective of nuclear factor-κB activation

Lipopolysaccharide (LPS) is an endotoxin involved in septic shock syndrome and potentiates toxicant-induced liver injury. The effects of LPS on the constitutive and inducible expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) genes were studied in rats. Northern blot analysis showed that treatment of rats with LPS caused suppression in mEH and GST gene expression. The mEH mRNA level was decreased in a time-dependent manner following a single dose of LPS (1 mg/kg, i.v.), resulting in levels of 52%, 22%, 17%, and 94% of those in untreated animals at 2, 6, 12, and 24 hr, respectively. The levels of rGSTA2 and rGSTA3 mRNA were suppressed in response to an LPS injection to the similar extents as observed in mEH mRNA, whereas rGSTM1 and rGSTM2 mRNA levels were less affected. LPS inhibited mEH gene expression at the doses of 1 μg or greater. Whereas treatment of rats with allyl disulfide (ADS), oltipraz (OZ) or pyrazine (PZ) at the dose of 50 mg/kg caused increases in the mEH mRNA level at 12 hr, a concomitant LPS injection (1 mg/kg) resulted in 80%–95% suppression of the inducible gene expression. The inducible rGSTA2, rGSTA3, rGSTM1, and rGSTM2 mRNA levels were also 50%–90% decreased at 12 hr after LPS treatment, with the relative change in rGSTA being greater than that in rGSTM. Three consecutive daily treatments with LPS (10 μg/kg/day) resulted in significant decreases of the constitutive and PZ (50 mg/kg/day, i.p. for 3 days)-inducible mEH and GST mRNA levels, which were consistent with those in the protein levels. Gel shift retardation analysis showed that LPS substantially activated the hepatic nuclear p65/p50 nuclear factor-κB (NF-κB) complex with the maximal effect observed at 1 hr at the doses of 1 μg/kg or greater. LPS-induced activation of nuclear NF-κB (1 μg/kg, i.v.) failed to be inhibited by concomitant treatment with the mEH and GST inducers, including ADS (300 mg/kg, p.o.), OZ (300 mg/kg, p.o.), and PZ (300 mg/kg, i.p.), indicating that NF-κB activation was not required for suppression of the gene expression by LPS. In contrast, GdCl 3, an inhibitor of mEH and GST expression, inhibited LPS-induced activation of the p65/p50 NF-κB. These gel shift analyses provided evidence that LPS-induced activation of the NF-κB was not responsible for alterations in the gene expression. In summary, the results of this research demonstrate that LPS effectively inhibits constitutive and inducible mEH and GST expression with decreases in their mRNA levels, and that LPS suppression in the expression of the detoxifying enzymes is not mediated with its activation of NF-κB.

Correlation of increased mortality with the suppression of radiation-inducible microsomal epoxide hydrolase and glutathione S-transferase gene expression by dexamethasone: effects on vitamin C and E-induced radioprotection

Previous studies in this laboratory have shown that γ-ray ionizing radiation in combination with oltipraz, a radioprotective agent, enhances hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) expression. The present study was designed to investigate the effects of dexamethasone on the radiation-inducible expression of mEH and rGST genes and on the vitamin C and E-induced radioprotective effects in association with the expression of the genes. Treatment of rats with a single dose of dexamethasone (0.01–1 mg/kg, p.o.) caused a dose-dependent decrease in the constitutive mEH gene expression at 24 hr. The radiation-inducible mEH mRNA level (threefold increase after 3 Gy γ-irradiation) was decreased by 21% and 88% by dexamethasone at the doses of 0.1 and 1 mg/kg, respectively. Although dexamethasone alone caused 2- to 5-fold increases in the hepatic rGSTA2 mRNA level, rats treated with dexamethasone prior to 3 Gy irradiation exhibited 80%–93% suppression in the radiation-inducible increases in the rGSTA2 mRNA level. The inducible rGSTA3 and rGSTA5 mRNA levels were also significantly decreased by dexamethasone, whereas the rGSTM1 mRNA level was reduced to a lesser extent. Vitamin C and/or E, however, failed to enhance the radiation-inducible increases in hepatic mEH and rGST mRNA levels. Whereas rats exposed to 3 Gy irradiation with or without vitamin C treatment (30 or 200 mg/kg/day, p.o., 2 days) exhibited ∼threefold increases in the mEH and rGSTA2/3/5 mRNA levels relative to untreated animals, dexamethasone treatment (1 mg/kg, p.o.) resulted in 64%–96% decreases in the mRNA levels at 24 hr. The inducible rGSTM1/2 mRNA levels in the vitamin C/E-treated rats were ∼50% suppressed by dexamethasone. Although vitamin C and/or E treatment (200 mg/kg/day, p.o., 2 days) improved the 30-day survival rates of the 8 Gy γ-irradiated mice from 39% up to 74%, the improved survival rate of γ-irradiated animals was reduced to 30% by dexamethasone pretreatment (1 mg/kg/day, 2 days). The mean survival time of dexamethasone-treated animals was reduced to ∼2 days from 14 days in the animals with total body irradiation alone. No significant hematologic changes were observed in mice at 10 days after dexamethasone plus γ-irradiation, as compared with irradiation alone. These results demonstrate that: dexamethasone substantially suppresses radiation-inducible mEH, rGSTA and rGSTM expression in the liver; vitamins C/E exhibit radioprotective effects without enhancing radiation-inducible mEH and GST gene expression; and inhibition of radiation-inducible mEH and rGST gene expression in the vitamin C- and E- treated animals by dexamethasone was highly correlated with reduction in the survival rate and the mean survival time of γ-irradiated animals.

Differential induction of rat hepatic microsomal epoxide hydrolase and rGSTA2 by diazines: the role of cytochrome P450 2E1-mediated metabolic activation

Previous studies have shown that pyridazine (PD) and pyrazine (PZ) are efficacious in inducing microsomal epoxide hydrolase (mEH) in the liver with elevation of the mRNA level. The present study was designed to investigate the expression of mEH and rGSTA2 genes in response to the diazines including PD, PZ and pyrimidine (PM) and the basis for their enzyme induction. Rats treated with either PD or PZ for 3 days resulted in marked increases in mEH and rGSTA2 mRNA levels with concomitant induction of the proteins, whereas PM failed to elevate the mRNA levels. Treatment of rats with a single dose of PD or PZ showed dose-dependent increases in mEH and rGSTA2 mRNA levels at 24 h with ED 50 values being ∼10 mg/kg. Time-course studies showed that the mRNA levels were increased to maximal extents at 24–48 h after treatment. Studies were extended to assess the mechanistic basis for the enzyme induction by PD and PZ. β-Naphthoflavone (BNF) caused a 6-fold increase of rGSTA2 mRNA in the liver (100 mg/kg per day, p.o., 3 days), as compared to control, whereas the agent failed to increase mEH mRNA level. Administration of PD or PZ (50 mg/kg) to BNF-pretreated rats resulted in no enhanced increase of the mEH mRNA as compared to the individual treatment, while the rGSTA2 mRNA level was additively elevated, suggesting the possibility that increases of the mEH and rGSTA2 mRNAs by PD or PZ might be mediated with antioxidant responsive element(s) in the genes, but not with xenobiotic responsive element. Western blot analysis revealed that cytochrome P450 2E1 was induced 3- to 4-fold by both PD and PZ, whereas PM failed to induce P450 2E1. Concomitant treatment of rats with PD or PZ in combination with acetone, a substrate for P450 2E1, caused no significant increase in the mEH and rGSTA2 mRNA levels relative to that in untreated animals, whereas PD or PZ treatment without a concomitant acetone administration resulted in marked increases of the mRNAs. Diazine-inducible mEH and rGSTA2 mRNA levels were ∼2-fold enhanced in P450 2E1-induced starved rats, as compared to those in diazine-treated unstarved animals. These data indicate that P450 2E1-mediated bioactivation of the diazines might contribute to transcriptional activation of the mEH and GST genes. These results provide evidence that both PD and PZ efficaciously induce mEH and rGSTA2 in the liver with increases in the mRNA levels, while PM is ineffective, and that induction of mEH and rGSTA2 may be mediated through bioactivation of the diazines by P450 2E1.

Evaluation of octamethylcyclotetrasiloxane (D4) as an inducer of rat hepatic microsomal cytochrome P450, UDP-glucuronosyltransferase, and epoxide hydrolase: a 28-day inhalation study.

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Enhancement of Radiation-Induced Hepatic Microsomal Epoxide Hydrolase Gene Expression by Oltipraz in Rats

The effects of radiation exposure in conjunction with oltipraz, a chemopreventive agent, on the expression of the gene encoding hepatic microsomal epoxide hydrolase (mEH) were examined in rats. Rats exposed to a single dose of 3 Gy gamma rays exhibited timerelated changes in the hepatic mEH mRNA level. Whereas the mEH mRNA level was transiently decreased at 3 and 8 h after irradiation, the mRNA levels were increased 3- to 4-fold at 15 to 48 h postirradiation, returning to the level in untreated animals at 72 h. Treatment of rats with oltipraz resulted in 1- to 19-fold increases in hepatic mEH mRNA levels 24 h post-treatment at doses of 5-200 mg/kg. Although treatment with oltipraz at a dose of 30 mg/kg affected the mEH mRNA level minimally (i.e. approximately 2-fold), 3 Gy whole-body irradiation along with oltipraz treatment resulted in a 9-fold increase in the mEH mRNA level at 24 h post-treatment. Treatment of animals with both oltipraz and 3 Gy gamma radiation for 3 consecutive days resulted in a 7-fold increase in mEH mRNA, showing that the increases in mEH mRNA were enhanced by the combination treatment. In rats irradiated with 3 Gy for 5 consecutive days, however, the mEH mRNA level failed to increase due to cell injury. Studies were further designed to assess the effects of 0.5 Gy ionizing radiation and concomitant oltipraz treatment. RNA blot analysis showed that mEH mRNA levels failed to be significantly altered at 3, 8, 15, 24 and 48 h after a single dose of 0.5 Gy. Nonetheless, exposure of animals to 0.5 Gy daily for 3 to 5 consecutive days caused a 3-fold elevation in the hepatic mEH mRNA level. Furthermore, treatment of animals with both oltipraz (30 mg/kg/day) and 0.5 Gy of gamma rays resulted in an enhanced elevation in the mEH mRNA level at 24 h post-treatment compared to the individual treatment, resulting in a 7-fold relative increase. The enhanced expression of hepatic mEH mRNA by 0.5 Gy gamma radiation and oltipraz was also observed after treatment for 3 to 5 days (8- to 6-fold relative increases). Western immunoblot analyses showed that hepatic microsomes produced from the rats treated with 0.5 Gy daily for 3 to 5 days resulted in a approximately 2-fold induction of hepatic mEH and that rats exposed to radiation in combination with oltipraz showed 3-fold increases in the liver mEH protein. Thus the relative increase in mEH mRNA levels was consistent with the expression of the protein. These results demonstrate that ionizing radiation causes alterations in hepatic mEH gene expression with the induction of the protein and that the mEH gene expression is enhanced by oltipraz treatment.

Enhancement of radiation-inducible hepatic glutathione-S-transferases Ya, Yb1, Yb2, Yc1, and Yc2 gene expression by oltipraz: possible role in radioprotection.

Previous studies have shown that radiation in combination with oltipraz enhances hepatic microsomal epoxide hydrolase expression. The effects of gamma-ray radiation exposure in combination with oltipraz on the expression of hepatic glutathione-S-transferase (GST) subunits Ya, Yb1, Yb2, Yc1, and Yc2 were examined in the rat. Northern RNA blot analyses revealed that GST mRNA levels were altered in response to daily 3- or 0.5-Gy doses of radiation. The hepatic GST mRNA levels were transiently decreased at 3 and 8 hr after a single 3-Gy dose of radiation. The GST Ya, Yb1, Yb2, Yc1, and Yc2 mRNA levels were increased by 2-4-fold at 15 and 24 hr after irradiation with 3 Gy, followed by return to the levels of untreated rats at 48 hr after treatment. The treatment of animals with oltipraz alone resulted in dose-related increases in the GST Ya, Yb1, Yc1, and Yc2 mRNA levels, whereas Yb2 mRNA levels were, minimally increased. Although a single dose of oltipraz (30 mg/kg orally) caused a minimal 2-fold elevation in the hepatic GST Ya mRNA level, exposure of animals to both oltipraz and 3-Gy radiation resulted in a 4-fold relative increase in GST Ya mRNA level, indicating that the Ya mRNA expression was additively enhanced by the combination treatment. The Yb1/2 and Yc1/2 mRNA expressions were also enhanced by oltipraz in combination with radiation. Multiple exposure of rats to daily 0.5-Gy radiation caused time-related increases in GST gene expression. The greatest enhancement in GST expression was observed at 24 hr after a single 0.5-Gy dose of radiation in conjunction with oltipraz (e.g., a 9-fold relative increase in GST Ya), whereas the relative additive increases in GST mRNA were less pronounced at day 3 or 5 after treatment. These increases in the GST mRNA levels were consistent with those in the immunochemically detectable GST protein levels. Histopathological examinations revealed that exposure of rats to radiation (0.5 Gy/day for 3-5 days) caused mild-to-moderate hepatocyte degeneration with sinusoidal congestion, whereas oltipraz (30 mg/kg/day for 3 days) was effective in blocking the radiation-induced liver injury. The enhanced expression of these GST isoforms by oltipraz may be associated in part with its hepatoprotective effect against the injury caused by ionizing radiation.

Differential induction of hepatic microsomal epoxide hydrolase by alkyl sulphides and alkyl ethers in rat

1. Previous studies have shown that the expression of certain cytochromes P450 and major glutathione S-transferases (GSTs) is differentially modulated by alkyl sulphide treatment. The current study was designed to examine the influence of several alkyl sulphides and alkyl ethers on the induction of hepatic microsomal epoxide hydrolase (mEH) in rat. 2. Northern blot analysis has shown that treatment of rats with allyl disulphide (ADS) or allyl sulphide (AS) (50 mg/kg/day, 7 days) resulted in 5-6-fold increases in mEH mRNA levels, whereas allyl ether failed to stimulate mEH gene expression. Rats treated with propyl disulphide (PDS) or propyl sulphide (PS) exhibited increases in mEH mRNA levels, although the relative increases were less than those produced by ADS or AS. Propyl ether failed to increase hepatic mEH mRNA levels. Immunoblot analyses confirmed that allyl and propyl sulphides induce hepatic mEH protein, with the agents containing allyl moiety being more effective. 3. Treatment of rats with ADS at 100 mg/kg resulted in 9-14-fold increases in mEH mRNA levels at 12 and 24 h post-treatment respectively, whereas AS caused a maximal increase at the 24-h time point. Animals treated with PDS or PS exhibited time-related increases in mEH mRNA levels, resulting in 2-6-fold elevations at 72-h post-treatment. The changes in mEH mRNA levels after ADS or AS treatment appeared to be more rapid than those after PDS or PS. The rank order for increases in mEH mRNA levels was ADS > AS > PS > PDS. The result of immunoblot analysis was consistent with that of Northern blot analysis. 4. Rats treated with isopropyl sulphide (IPS),butyl sulphone, tert-butyl sulphide,secbutyl sulphide or butyl sulphide (100 mg/kg/day) showed 3-5-fold increases in the mEH mRNA levels at 3 days post-treatment. IPS treatment caused a similar increase in the mRNA level to that of PS, whereas the induction efficacy of PDS was lower than that of PS or IPS. Treatment with butyl sulphone as well as the structural isomers of butyl sulphides caused approximately 3-fold elevations in mEH mRNA levels. 5. These results provide evidence that alkyl sulphides, but not alkyl ethers, are capable of increasing mEH mRNA levels and that the organosulphur compounds with the allyl moiety are more effective in stimulating the mEH gene than are saturated alkyl sulphides. The comparable increase in mEH mRNA by butyl sulphone as well as the necessity of sulphur atom suggests that sulphone metabolites from the alkyl sulphides might be the ultimate inducers of mEH.

Localization of microsomal epoxide hydrolase in normal and neoplastic human kidney.

Microsomal epoxide hydrolase is a xenobiotic metabolizing enzyme that catalyzes the conversion of toxic and carcinogenic epoxides to less toxic dihydrodiols. The cellular localization and distribution of microsomal epoxide hydrolase were investigated for the first time in normal and neoplastic human kidney. Light microscopic immunohistochemical studies using an alkaline phosphatase-anti-alkaline phosphatase technique showed that in normal kidney there was a wide distribution of epoxide hydrolase immunoreactivity. The main localization of epoxide hydrolase immunoreactivity was to the proximal and distal tubule epithelial cells. Strong epoxide hydrolase immunoreactivity was also identified in epithelium of the collecting ducts. In addition, epoxide hydrolase immunoreactivity was present in vascular endothelial cells, including endothelial cells lining glomerular capillaries. Epoxide hydrolase immunoreactivity was identified in all the renal tumors, and in each tumor immunoreactivity for epoxide hydrolase was localized to tumor cells. Immunoblotting of both normal kidney and tumor microsomes confirmed the presence of a single protein band of molecular weight 49 KD corresponding to the molecular weight of human hepatic microsomal epoxide hydrolase.

Induction of rat liver microsomal epoxide hydrolase by its endogenous substrate 16 alpha, 17 alpha-epoxyestra-1,3,5-trien-3-ol.

1. The influence of the endogenous steroid epoxides 16 alpha, 17 alpha-epoxyestra-1,3,5(10)-trien-3-ol (estroxide) and 16 alpha, 17 alpha-expoxiandrost-4-en-3-one (androstene oxide) and their metabolic precursors estra-1,3,5(10), 16-tetraen-3-ol (estratetraenol) and androsta-4, 16-dien-3-one (androstadienone) on the specific activities of hepatic microsomal and soluble epoxide hydrolase, glutathione S-transferase, dihydrodiol dehydrogenase, and 7-ethoxycoumarin deethylase was investigated in the male Sprague-Dawley rat. 2. Both estroxide and estratetraenol induced microsomal epoxide hydrolase activity towards styrene oxide and estroxide itself 2-2.5-fold and glutathione conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) 1.6-fold after intraperitoneal administration of a high dose of compound (300 mg per kg of body weight). 3. In addition, estroxide decreased 7-ethoxycoumarin deethylation down to 20% of the activity observed in the untreated rat, whereas estratetraenol enhanced the activity of soluble epoxide hydrolase towards trans-stilbene oxide by a factor of 1.7. 4. In contrast, neither androstene oxide nor androstadienone showed a significant influence on any of the parameters under investigation. Dihydrodiol dehydrogenase was not significantly changed by any of the treatments.

1-Benzylimidazole induces rat hepatic microsomal epoxide hydrolase with the elevation of its mRNA levels.

The effects of 1-benzylimidazole on the expression of microsomal epoxide hydrolase (mEH) gene were examined in rat. Immunoblot analyses showed that hepatic microsomes isolated from rat treated with 1-benzylimidazole at 25, 50 or 100 mg/kg/day for 3 or 6 days exhibited 2-3-fold increases in mEH levels, relative to control microsomes. Northern and slot blot analysis revealed that 1-benzylimidazole is effective in increasing hepatic mEH mRNA levels at 24 h post-treatment (i.e. 4-5-fold). Hepatic mEH mRNA levels were elevated from 4-4- to 8-fold following 1-benzylimidazole administration in a time-dependent manner. These results demonstrated that 1-benzylimidazole induces mEH protein in rat and that the induction is primarily associated with accumulation of mEH mRNA levels.