mEH mRNA Levels Research Articles

Inhibition of human m-epoxide hydrolase gene expression in a case of hypercholanemia

Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in carcinogen metabolism and is also able to mediate the sodium-dependent uptake of bile acids into hepatocytes. Studies have identified a subject (S-1) with extremely elevated serum bile salt levels in the absence of observable hepatocellular injury, suggesting a defect in bile acid uptake. In this individual, mEH protein and mEH mRNA levels were reduced by approximately 95% and 85%, respectively, whereas the expression and amino acid sequence of another bile acid transport protein (NTCP) was unaffected. Sequence analysis of the mEH gene (EPHX1) revealed a point mutation at an upstream HNF-3 site (allele I) and in intron 1 (allele II), which resulted in a significant decrease in EPHX1 promoter activity in transient transfection assays. Gel shift assays using a radiolabeled oligonucleotide from each region resulted in specific transcription factor binding patterns, which were altered in the presence of the mutation. These studies demonstrate that the expression of mEH is greatly reduced in a patient with hypercholanemia, suggesting that mEH participates in sodium-dependent bile acid uptake in human liver where its absence may contribute to the etiology of this disease.

Activation of phosphatidylinositol 3-kinase by oxidative stress leads to the induction of microsomal epoxide hydrolase in H4IIE cells

We have shown that PI3-kinase played an essential role in the ARE-mediated rGSTA2 induction by oxidative stress following SAAD (Mol. Pharmacol. 58 (2000) 1017). Microsomal epoxide hydrolase (mEH), which detoxifies a variety of epoxide intermediates produced from various xenobiotics, is inducible by oxidative stress. In the present study, we studied whether sulfur amino acid deprivation (SAAD) activated phosphatidylinositol 3-kinase (PI3-kinase)/Akt and induced mEH in H4IIE cells. The role of PI3-kinase activation on the mEH induction by SAAD was also investigated. PI3-kinase was activated from 10 min through 12 h after SAAD, the activity of which returned to control level at 24 h. The activation of PI3-kinase led to increases in the activity of Akt at the same time points. Northern and Western blot analyses revealed that the mEH mRNA level was four-fold increased at 48 h, which accompanied the induction of mEH protein. Wortmannin or LY294002, PI3-kinase inhibitors, completely inhibited the increases in mEH mRNA and protein by SAAD. These results demonstrated that SAAD activated the PI3-kinase/Akt pathway at early stages and induced mEH, presumably as an adaptive response, and that the PI3-kinase/Akt pathway played a crucial role in the induction of mEH.

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.

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.

Partial hepatoprotective effects of allylthiobenzimidazole in the absence of cytochrome P4502E1 suppression: effects on epoxide hydrolase, rGSTA2, rGSTA3/5, rGSTM1 and rGSTM2 expression

1. 2-(Allylthio)pyrazine protects the liver against acetaminophen- and carbon tetrachloride-induced injury through inhibition of cytochrome P4502E1 and induction of glutathione S -transferases (GSTs). By comparison, the effects of allylthiobenzimidazole (ATB) on the levels of several hepatic cytochrome P450, microsomal epoxide hydrolase (mEH) and GST expression have been studied in the rat herein. 2. Western immunoblotting analyses revealed that ATB treatment (50 mg/kg/day for 5 days) failed to alter cytochrome P4501A2, P4502B1/2 and P4502E1 levels in the liver, whereas the expression of P4502C11 was reduced ∼50% by ATB. 3. Treatment of rat with a single dose of ATB resulted in 2-21-fold increases in mEH mRNAlevels at 24 h withan ED50 = 60 mg/kg. mEH mRNAlevelwas elevated 9- and 21- fold at 12 and 24 h after treatment at 200 mg/kg respectively as compared with control. Western blot analysis revealed that ATB induced mEH protein levels by 2-fold relative to control. 4. ATB induced the major GST mRNA levels as a function of dose, resulting in rGSTA2, rGSTA3/5 and rGSTM1 mRNA levels elevated by 20-, 6- and 8-fold at 24 h respectively. The relative rGSTM2 mRNA level was minimally affected. Time-course studies showed that mEH, rGSTA2 and rGSTM1 mRNA levels were significantly increased at 12 and 24 h after ATB treatment, returning to control levels by 48 h. Treatment of rat with ATB (20-50 mg/kg/day for 5 days) resulted in 2-3-fold increases in mEH, rGSTA1/2, rGSTA3/5 and rGSTM1 mRNA levels with the induction of GST subunits. 5. ATB failed to block carbon tetrachloride-induced liver toxicity in rat and mouse. ATB treatment (50 mg/kg day for 3 days) prior to a lethal dose of acetaminophen significantly reduced acetaminophen-induced liver toxicity in mouse, as assessed by both plasma alanine aminotransferase activity and histopathological examination. The 30-day survival rate of mouse γ-irradiated at 8 Gy failed to be improved by ATB pretreatment (100 mg/kg/day for 2 days). 6. These results provided evidence that ATB stimulated mEH and GST gene expression at early times and reduced the P4502C11 level in the absence of P4502E1 suppression. ATB was only partially effective in protecting the liver against toxicant induced injury despite the presence of allylthio moiety in its chemical structure.

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.

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.

Expression of glutathione S-transferases Ya, Yb1, Yb2, Yc1 and Yc2 and microsomal epoxide hydrolase genes by thiazole, benzotbiazole and benzothiadiazole

The effects of thiazole (TH), benzothiazole (BT) and benzothiadiazole (BZ) on the expression of hepatic glutathione S-transferases (GSTs) Ya, Yb1, Yb2, Yc1 and Yc2 and microsomal epoxide hydrolase (mEH) genes were compared in rats. TH treatment resulted in 4- to 24-fold increases in GST Ya mRNA levels at 24 hr posttreatment; the ED 50 value was 70 mg/kg. GST Ya mRNA levels were elevated 13-, 20-, 20- and 9-fold at 12, 24, 48 and 72 hr following 100 mg/kg of TH treatment, respectively, as compared with the control. BT was a moderate inducer of GST Ya with a maximal 18-fold increase observed, whereas BZ treatment caused a transient increase in GST Ya mRNA at 12 hr posttreatment, followed by a return to a 4-fold relative increase at 24 hr or afterward. Treatment of rats with TH at the dose of 100 mg/kg resulted in an ∼10-fold increase in either Yb1 or Yb2 mRNA levels at 24 hr posttreatment. BT-treated rats showed 7- and 3-fold increases in the GST subunit Yb1 and Yb2 mRNA levels at 24 hr posttreatment. BZ was the least effective in modulating either GST Ybl or Yb2 mRNA, resulting in < 2-fold changes. GST Yc1 and Yc2 mRNA levels were increased ∼8-fold at the dose of 200 mg/kg of TH. BT minimally affected GST subunit Yc1 and Yc2 mRNA levels, with a maximal 4-fold relative increase observed. BZ was the least effective in enhancing Yc1 and Yc2 mRNA levels. Protein levels for GST subunit Ya, Yb1, Yb2 and Yc were also elevated in response to TH by 3-, 2-, 2- and 2-fold, respectively. Thus, TH was effective in modulating both constitutive and inducible GST gene expression. BT or BZ was much less effective in increasing the expression of GST subunits. These RNA and Western blot analyses revealed that the levels of major GST were differentially increased after treatment with these thiazoles, exhibiting a rank order of GST expression of TH > BT > BZ. mEH expression by these compounds appeared to be consistent with that of GST Ya. The mRNA levels for GST Ya, Yb1, Yb2, Yc1 and Yc2 and mEH were also determined after treatment with triazole (TR), imidazole (IM), benzoxazole (BX), benzotriazole (BTR) or benzimidazole (BIM). TR, IM, BX or BTR caused increases in Ya, Yb1, Yc1 and Yc2 mRNA levels by 2- to 3-fold, whereas the agents failed to modulate the expression of GST Yb2. The fact that benzene, cyclohexane or n-hexane minimally affected the major GST or mEH mRNA levels provided evidence that certain heterocyclic compounds are more capable of modulating GST or mEH gene expression than hydrocarbons. These results corroborate evidence that the thiazoles differentially stimulate GST or mEH genes, with TH being the most efficacious; that thiazoles with carbocyclic ring are much less effective in increasing GST or mEH levels than is TH; and that the changes in these GST and mEH levels are primarily associated with increases in mRNA levels.

Expression of rat microsomal epoxide hydrolase during pregnancy

Microsomal epoxide hydrolase (mEH) protein and messenger ribonucleic acid (mRNA) levels were assessed in maternal rats during pregnancy and mEH gene expression was compared with cytochrome P450 expression. Immunoblot analysis using goat anti-rat mEH antibody showed that hepatic mEH levels in adult rats at day 12 of gestation were comparable to those in virgin female rats at the same age, whereas mEH protein levels were substantially decreased by ~70% at day 20 of pregnancy. Northern and slot blot analyses revealed that hepatic mEH mRNA levels failed to be modulated at day 12 of pregnancy, whereas mEH mRNA levels were decreased to ~20% of virgin control in late pregnancy ( i.e. day 20), which was consistent with the result of mEH immunoblot analysis. Hepatic cytochrome P4502E1 levels were also diminished at day 12 and day 20 of gestation by ~50% and ~70%, respectively, relative to virgin control rats, as supported by immunoblot analysis. Hepatic P4502E1 mRNA levels at day 12 and day 20 of pregnancy were also significantly decreased to 30% and 8% of virgin rats at 17 weeks of age, respectively, demonstrating that suppression in P4502E1 protein levels accompanied decreases in its mRNA expression. The levels of cytochrome P4501A2 mRNA at either day 12 or day 20 of pregnancy was decreased by ~50% relative to virgin female rats, which was less than that observed in mEH or P4502E1 expression. Pregnancy failed to affect P4501A1 mRNA levels, which were not detectable in female rats at 17 weeks of age. Renal mEH mRNA levels in maternal rats at day 20 of gestation were also decreased to 25% of virgin control. These results demonstrated that levels of mEH and P4502E1 proteins decreased significantly in late pregnancy with concomitant decreases in their mRNA levels.

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.

Differential expression of microsomal epoxide hydrolase gene by azole heterocycles in rats

The effects of heterocycles including imidazole (IM), 1,2,4-triazole (TR) and thiazole (TH) on the expression of microsomal epoxide hydrolase (mEH) gene were examined in rats (200 mg kg body weight day , i.p.). Hepatic microsomes prepared from rats treated with IM for 3 days failed to exhibit an increase in mEH protein level whereas TR treatment resulted in an approximately 2- to 3- fold levation in hepatic mEH levels relative to control, as assessed by both SDS-PAGE and immunoblot analyses. In contrast, thiazole-induced hepatic microsomes resulted in a substantial increase in mEH levels (i.e.∼5-fold). Slot and northern blot analyses, probed with an mEH cDNA, showed that the hepatic mEH mRNA levels in the animals treated with IM for 3 days were marginally increased by ∼2-fold, as compared with untreated animals, whereas TR caused an ∼8-fold increase in hepatic mEH mRNA levels after three consecutive daily treatments. TH treatment resulted in an ∼22-fold increase in the mEH mRNA levels, demonstrating that TH is the most efficacious among these three azole heterocycles. Because TH was the most effective in increasing hepatic mEH protein and mRNA levels, the agent was chosen for further evaluation. Time course of mEH gene expression at early times after a single treatment with TH was determined and compared with that caused by pyrazine (PZ), a strong mEH inducer. Hepatic mEH mRNA levels were increased ∼1-, 3-, 20- and 16-fold at 3, 6, 12 and 24 hr, respectively, following TH treatment, relative to control, whereas mEH mRNA levels were elevated ∼1-, 1-, 22- and 18-fold, respectively, at the same time points after PZ treatment, as monitored by slot RNA hybridization analyses. Northern blot analyses using either total RNA or poly(A) + RNA fractions exhibited comparable time courses in increasing mEH mRNA levels after TH or PZ treatment with maximal mRNA increases being noted at 12 hr post treatment. Although neither IM or TR failed to affect renal mEH gene expression to a notable extent, TH treatment caused 6- to 8-fold increases in kidney mEH mRNA levels, with a 2-fold increase in mEH protein detected. These results demonstrated that the azole heterocyclic compounds IM, TR and TH differentially induce mEH with TH as the most efficacious azole; and that the changes in mEH levels are primarily associated with increases in mRNA levels.

Enhanced expression of rat microsomal epoxide hydrolase gene by organosulfur compounds

The effects of organosulfur compounds including allylsulfide (AS), allylmereaptan (AM) and allylmethylsulfide (AMS) on the expression of microsomal epoxide hydrolase (mEH) protein and its mRNA were examined in rats. The levels of mEH induction were examined with or without concomitant treatment of animals with pyrazine, a strong inducer of mEH, in order to establish whether a common molecular basis exists for mEH induction between these structurally different xenobiotics. Immunoblot analyses using anti-rat mEH antibody showed that treatment with AS caused an ∼4-fold increase in hepatic mEH protein levels relative to controls whereas treatment with both AS and pyrazine resulted in only minimal additive increases in the elevation of mEH. Administration of AM to rats resulted in a comparable increase in mEH levels to that caused by AS, whereas an ∼2-fold increase was noted after AMS treatment, as compared to control. mEH levels in the hepatic microsomes isolated from animals treated with both AMS and pyrazine were, however, ∼50% less than those from pyrazine-treated rats. Thus, AS and AM appeared to be more effective than AMS in elevating mEH, as evidenced by immunoblot analyses. The levels of mEH mRNA were increased 10–16-fold following treatment with either AS or AM, while AMS caused a 3–7-fold increase relative to control, as assessed by slot blot analysis probed with a 1.3 kb mEH cDNA. Time-dependent increases in mRNA levels by each of these organosulfur compounds were consistent with those in mEH protein levels at 3 days. A marginal additive increase in mEH mRNA levels was noted following co-administration of either AS or AM with pyrazine, whereas treatment with both AMS and pyrazine decreased mEH mRNA levels by 55%. Significant mEH mRNA increases in poly (A) + RNA fractions were confirmed by northern blot analysis. The results demonstrate that these organosulfur compounds are inducers of mEH and that the induction involves increases in its mRNA.