Expression Of Microsomal Epoxide Hydrolase Research Articles

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.

Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats.

The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and also in vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrine N-demethylase and ethoxyresorufin O-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal epoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication of DL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.

Expression of xenobiotic metabolizing enzymes in breast cancer.

We have studied the expression of different xenobiotic metabolizing enzymes in primary operable breast cancer of no special type. The expression of two forms of cytochrome P450, microsomal epoxide hydrolase, and three classes of glutathione S-transferase was investigated using immunohistochemistry. The tumours were characterized by consistent expression of microsomal epoxide hydrolase and by variable expression of the two forms of cytochrome P450 and the three types of glutathione S-transferase. Cytochrome P450 1A and cytochrome P450 3A were identified in 39 and 22 per cent of tumours, respectively. In each case, immunostaining was present only in areas of invasive carcinoma. Epoxide hydrolase was identified in 89 per cent of tumours and glutathione S-transferases pi, mu, and alpha were identified in 56, 65, and 44 per cent of tumours, respectively. Immunoreactivity for epoxide hydrolase and glutathione S-transferases was identified in both tumours and non-neoplastic breast tissue. The presence of different xenobiotic metabolizing enzymes may have a role in determining the intrinsic drug resistance of breast cancer to a variety of anti-cancer drugs, and the expression of these enzymes can readily be assessed using immunohistochemistry.