Expression Of Microsomal Epoxide Hydrolase Research Articles

Tissue-, Region-, and Gene-Specific Induction of Microsomal Epoxide Hydrolase Expression and Activity in the Mouse Intestine by Arsenic in Drinking Water.

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.

Evaluation and clinical implications of interactions between compound Danshen dropping pill and warfarin associated with the epoxide hydrolase gene.

Introduction: In clinical practice, warfarin is often combined with Compound Danshen dripping pill (CDDP) for the treatment of cardiovascular diseases. However, warfarin has a narrow therapeutic index, wide interindividual variability (genetic and non-genetic factors), and is susceptible to drug-drug interactions. Our previous study indicated that CDDP might interact with warfarin in individuals with the epoxide hydrolase gene (EPHX1; single-nucleotide polymorphism: rs2292566) A/A subtype. We sought to clarify the interaction between CDDP and warfarin associated with EPHX1 in a comprehensive and accurate manner. Methods: Here, EPHX1 A and EPHX1 G cell lines were established. Expression of microsomal epoxide hydrolase (mEH), vitamin K epoxide reductase (VKOR), and vitamin K-dependent clotting factors (FII, FVII, FIX, FX) was measured by western blotting upon incubation with CDDP and warfarin. mEH activity was evaluated by measuring the transformation of epoxyeicosatrienoic acids into dihydroxyeicosatrienoic acids. Then, healthy volunteers (HVs) with the EPHX1 A/A genotype were recruited and administered warfarin and CDDP to investigate the pharmacokinetics and pharmacodynamics of warfarin. Results: CDDP combined with warfarin could decrease expression of mEH and VKOR, and increase protein expression of FII, FVII, FIX, and FX, in EPHX1 A cells. CDDP could slightly influence the pharmacokinetics/pharmacodynamics of warfarin in HVs with the EPHX1 A/A genotype. Discussion: Rational combination of CDDP and warfarin was safe with no risk of bleeding, but the therapeutic management is also needed. The clinical study is posted in the China Clinical Trial Registry (ChiCTR190002434).

Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes

The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography–tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.

Low Microsomal Epoxide Hydrolase Expression is Associated with Bladder Carcinogenesis and Recurrence

Microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics and is associated with several forms of cancer. Here, we investigated the role of mEH expression in bladder carcinogenesis, subsequent progression and recurrence. The expression of mEH was analyzed by Western blot in 50 bladder urothelial carcinoma and 20 normal epithelial tissues. There was a significantly higher mEH expression in the normal epithelium (P<0.05) and mEH expression was lower in high stage than in low stage tumors (P<0.05). Further, immunohistochmistry in 106 bladder urothelial carcinoma demonstrated mEH expression to be negatively correlated with histological grade, pT stage and recurrence (P<0.05). These findings suggest the important role of mEH in bladder carcinogenesis, cancer development and recurrence, providing support for efforts to develop mEH-based gene therapy.

Ovarian expressed microsomal epoxide hydrolase: Role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P < 0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased ( P < 0.05) on day 4 of VCD (30 μM) exposure, followed by increased ( P < 0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH.

Microsomal epoxide hydrolase expression in the endometrial uterine corpus is regulated by progesterone during the menstrual cycle

We have shown previously that high expression levels of microsomal epoxide hydrolase (mEH) correlate with a poor prognosis of breast cancer patients receiving tamoxifen, suggesting that enhanced mEH expression could lead to antiestrogen resistance (Fritz et al. in J Clin Oncol 19:3-9, 2001). Thus, the purpose of this study was to gain insights into the role of mEH in hormone-responsive tissues. We analyzed biopsy samples of the endometrium by immunohistochemical staining, pointing to a regulation of mEH during the menstrual cycle: during the first half mEH expression was low, increased during the second half and reached highest levels during pregnancy. Additionally, the progesterone receptor (PR) positive human endometrial cell lines IKPRAB-36 (estrogene receptor alpha [ERalpha] negative) and ECC1-PRAB72 (ERalpha positive) were chosen to further investigate the hormonal regulation of mEH expression. Western Blot and quantitative RT-PCR analysis revealed an increase of mEH expression after treatment with medroxy-progesterone 17-acetate (MPA) in the ERalpha containing ECC1-PRAB72 cells. In contrast our results suggest that MPA had no influence on the mEH protein level in the ERalpha- IKPRAB-36 cells. In conclusion, mEH expression is regulated by progesterone in the presence of both PRs and ERalpha.

Effect of CYP2E1 Gene Deletion in Mice on Expression of Microsomal Epoxide Hydrolase in Response to VCD Exposure

Females are born with a finite number of primordial follicles. 4-Vinylcyclohexene diepoxide (VCD) is a metabolite formed by epoxidation of 4-vinylcyclohexene (VCH) via its two monoepoxides 1,2- and 7,8-4-vinylcyclohexene monoepoxide (VCM). VCD specifically destroys small preantral (primordial and small primary) follicles in the rodent ovary. The phase I enzyme, cytochrome P450 isoform 2E1 (CYP2E1) is involved in ovarian metabolism of VCM to VCD. Further, microsomal epoxide hydrolase (mEH) can detoxify VCD to an inactive tetrol (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). This study evaluated the effects of VCD-induced ovotoxicity on mEH in CYP2E1+/+ and -/- mice (129S(1)/SvImJ background strain) using a postnatal day 4 mouse whole ovary culture system. The hypothesis of our study is that there is a relationship between CYP2E1 and mEH gene expression in the mouse ovary. Relative to control, VCD exposure caused follicle loss (p < 0.05) in ovaries from both genotypes; however, after 15 days, this loss was greater (p < 0.05) in CYP2E1+/+ ovaries. In a time course (2-15 days), relative to control, VCD (5 microM) caused an increase (p < 0.05) in mEH mRNA by 0.5-fold (day 10) and 1.84-fold (day 15) in CYP2E1-/- but not +/+ ovaries. 7,12-Dimethylbenz[a]anthracene (DMBA) also destroys ovarian follicles but, unlike VCD, is bioactivated by mEH to an ovotoxic 3,4-diol-1,2-epoxide metabolite. Incubation of ovaries in increasing concentrations of DMBA (0.5-1 microM, 15 days) resulted in greater (p < 0.05) follicle loss in CYP2E1-/-, relative to +/+ ovaries. With greater mEH (CYP2E1-/-), increased follicle loss with DMBA (bioactivation) and decreased follicle loss with VCD (detoxification) support that ovarian expression of CYP2E1 and mEH may be linked.

Genetic polymorphism and gene expression of microsomal epoxide hydrolase in non-small cell lung cancer

Genetic polymorphisms of microsomal epoxide hydrolase (mEH) have been associated with increased risk of lung cancer. However, expression of mEH and its clinical significance in non-small cell lung cancer (NSCLC) have not been investigated. In this study we investigated the expression and genetic polymorphism of mEH in non-small cell lung cancer (NSCLC) patients. Genetic polymorphism was determined by restriction fragment length polymorphism of polymerase chain reaction (PCR) products. The allelic expression pattern as well as expression level of mEH were determined by reverse transcription-PCR (RT-PCR), cDNA sequencing, sequence alignment, immunoblotting and immunohistochemistry. Genotype distributions of mEH in Taiwan's NSCLC patients were 44.4% of 340TAC/340TAC, 48.6% of 340TAC/340CAC, and 7.0% of 340CAC/340CAC in exon 3, and 80.6% of 418CAT/418CAT, 19.4% of 418CAT/418CGT and 0% of 418CGT/418CGT in exon 4. Of the 72 NSCLC biopsies analyzed, mEH was expressed in 60 (83%) surgical specimens, and the major allelic expression pattern was fast type (Tyr113) in exon 3 (90.3%) and slow type (His139) in exon 4 (100%). Immunohistochemical staining showed that mEH was expressed in 326 of 423 (77.0%) tumor (lung tissue) specimens and in 48 of 93 (51.6%) metastatic lymph nodes. A significant difference in patient survival was found when mEH expression and adriamycin-containing chemotherapy were used to group patients (p=0.0167). In conclusion, with the combination of fast type (Tyr113) and slow type (His139), the mEH enzyme expressed in most NSCLC patients may have intermediate activity. Our findings indicate that with respect to cancer risk and disease progression, the expression level of mEH is as important as genetic polymorphism. In addition, mEH expression in NSCLC could be involved in drug resistance and prognosis of patients.

Expression of microsomal epoxide hydrolase is elevated in Alzheimer's hippocampus and induced by exogenous β‐amyloid and trimethyl‐tin

The brain is a potential target for drugs and environmental toxins. Microsomal epoxide hydrolase (mEH) is one of several critical biotransformation enzymes in xenobiotic metabolism and detoxification. In the present study, we report that the expression of mEH is significantly elevated in the hippocampus and associated cortex, but not in the cerebellum, in Alzheimer's disease (AD) patients. A large proportion of the mEH-positive cells are located around beta-amyloid plaques. The mEH-positive-staining cells are astrocytes and pyramidal neurons. Western blotting analysis confirmed increased expression of mEH in AD hippocampal tissues. In primary hippocampal glial culture, beta-amyloid aggregation stimulated mEH expression in the astrocytes, which displayed a patchy distribution. An environmental neurotoxic agent, trimethyl-tin, also activated mEH expression in rat hippocampus and entorhinal cortex. The present study demonstrates a significant increase in mEH expression in the AD hippocampus, a region showing abundant neuropathology in AD. The expression of mEH could also be elevated by exposure to exogenous beta-amyloid in vitro and environmental toxins in vivo. Our studies suggest that mEH may play a role in pathogenesis of neurodegeneration in response to environmental stress.

Accelerated Ovarian Failure Induced by 4-Vinyl Cyclohexene Diepoxide in Nrf2 Null Mice

Genetic and biochemical analyses have uncovered an essential role for nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating phase II xenobiotic metabolism and antioxidant response. Here we show that Nrf2 protects against the ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD) in mice. Nrf2-/- female mice exposed to VCD exhibit an age-dependent decline in reproduction leading to secondary infertility accompanied by hypergonadotropic hypogonadism after 30 weeks of age. VCD is shown to selectively destroy small ovarian follicles, resulting in early depletion of functional follicles. Treatment with VCD induces apoptotic death in cultured cells and in ovarian follicles, suggesting apoptosis as a mechanism of follicle loss. Loss of Nrf2 function blocks the basal and inducible expression of microsomal epoxide hydrolase, a key enzyme in the detoxification of VCD, and increases the oxidative stress in cells that is further exacerbated by VCD. Foxo3a, a repressor in the early stages of follicle activation, displays reduced expression in Nrf2-/- ovaries, causing accelerated growth of follicles in the absence of exposure to exogenous chemicals. Furthermore, Foxo3a is degraded through the 26S proteasome pathway in untreated cells and is induced by VCD via both Nrf2-dependent transcription and protein stabilization. This study demonstrates that Nrf2 serves as an essential sensor and regulator of chemical homeostasis in ovarian cells, protecting the cells from toxic chemicals by controlling metabolic detoxification, reactive oxygen species defense, and Foxo3a expression. In addition, these findings raise the possibility that exposure to environmental or occupational ovotoxicants plays a role in the premature ovarian failure commonly associated with infertility and premature aging in women.

Insulin and glucagon signaling in regulation of microsomal epoxide hydrolase expression in primary cultured rat hepatocytes.

Microsomal epoxide hydrolase (mEH) plays an important role in the detoxification of a broad range of epoxide intermediates and has been reported to be decreased during diabetes and fasting. The signaling pathways involved in the regulation of mEH expression in response to insulin and glucagon were examined in primary cultured rat hepatocytes. mEH protein levels were increased 2- to 6-fold in hepatocytes cultured for 1 to 4 days, respectively, in the presence of insulin. Concentration-response studies revealed that insulin concentrations >or=1 nM resulted in increased mEH protein levels. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated increase in mEH protein levels. The p38 mitogen-activated protein (MAP) kinase inhibitors SB203580 and SB202190 also abrogated the insulin-mediated increase in mEH protein. Treatment of cells with glucagon, 8-bromo-cAMP, or dibutyryl-cAMP for 3 days resulted in decreased mEH protein levels. Pretreatment with the protein kinase A (PKA) inhibitor H89 (N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline) prior to glucagon addition markedly attenuated the glucagon effect, implicating PKA signaling in the regulation of mEH expression. These data demonstrate that insulin and glucagon regulate, in an opposing manner, the expression of mEH in primary cultured rat hepatocytes. Furthermore, these data suggest that PI3K and p70 S6 kinase are active in the regulation of insulin-mediated mEH expression. We also provide data implicating p38 MAP kinase in the insulin-mediated increase in mEH levels. Moreover, cAMP and PKA are implicated in mediating the inhibitory effect of glucagon on mEH expression.

594 Preparation and use of peptide standards in measurements of 1,3-butadiene hemoglobin adducts

The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography–tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.

Microsomal epoxide hydrolase expression as a predictor of tamoxifen response in primary breast cancer: a retrospective exploratory study with long-term follow-up.

It has been suggested that estrogen receptor-independent high-affinity binding sites for antiestrogens could limit their local bioavailability and response. Microsomal epoxide hydrolase (mEH) was recently shown to be a component of the antiestrogen binding site complex. We investigated whether mEH expression in primary breast tumors is related to disease outcome and to the efficacy of tamoxifen treatment. Expression of mEH was semiquantitatively assessed by immunohistochemistry in sections prepared from archival paraffin blocks of primary breast cancers from 179 patients with a mean follow-up time of 81 months. Expression of mEH was correlated with poor disease outcome in all patients (P: < .01; n = 179) and in patients receiving tamoxifen (P: < .01; n = 78), but not in patients not treated with tamoxifen. Moreover, mEH was an independent prognostic factor by Cox regression analysis. The results of this first exploratory study suggest that mEH expression in primary breast cancer could be of predictive value for response to tamoxifen treatment and/or may be a novel independent prognostic factor for survival. The results are in agreement with the model that mEH participates in an estrogen receptor-independent tamoxifen- binding complex.

Distribution of microsomal epoxide hydrolase in humans: an immunohistochemical study in normal tissues, and benign and malignant tumours.

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.

Differential Protection by Rat UDP-Glucuronosyltransferase 1A7 against Benzo[a]pyrene-3,6-quinone- versus Benzo[a]pyrene-Induced Cytotoxic Effects in Human Lymphoblastoid Cells

UDP-glucuronosyltransferase 1A7 (UGT1A7) is a polyaromatic hydrocarbon (PAH)–inducible UGT with activity toward various benzo[a]pyrene (B[a]P) metabolites. To investigate the influence of rat UGT1A7 on B[a]P-induced cytotoxicity, human lymphoblastoid L3 cells were transfected with pMF6 (control expression vector), p167Dtk2 (microsomal epoxide hydrolase expression vector), or p167Dtk2-1A7 (epoxide hydrolase/UGT1A7 coexpression vector), and the cell populations were compared for sensitivity to B[a]P-induced effects. B[a]P inhibited cell proliferation and decreased relative cell survival of p167Dtk2 and p167Dtk2-1A7 cells to a similar extent. Metabolism studies using [3H]B[a]P revealed increased formation of glucuronide conjugates of B[a]P-4,5-diol, 3-OH-, or 9-OH-B[a]P and an unidentified metabolite by p167Dtk2-1A7 cells, but the presence of unconjugated metabolites suggested that glucuronidation capacity may be limited. No differences between p167Dtk2 and p167Dtk2-1A7 L3 cells were observed in the growth inhibitory effects of 3-OH-B[a]P or B[a]P-7,8-diol, but p167Dtk2-1A7-expressing cells were found to be less sensitive to B[a]P-3,6-quinone-induced effects on cell proliferation and relative cell survival. The effect was also observed in AHH-1 lymphoblastoid cells expressing UGT1A7 without epoxide hydrolase. The UGT1A7-expressing AHH-1 cells were also less sensitive to growth inhibition by B[a]P-1,6-quinone and B[a]P-6,12-quinone. Flow cytometric analysis of vehicle and B[a]P-3,6-quinone-exposed cell populations showed an association between UGT1A7 expression and resistance to B[a]P-3,6-quinone-induced apoptosis and loss of cell viability. These data suggest that UGT1A7 may be preferentially active toward B[a]P-quinones and that UGT1A7 may represent the PAH-inducible UGT activity previously implicated in protection against toxic redox cycling by B[a]P-3,6-quinone.

Role of gene knockout mice in understanding the mechanisms of chemical toxicity and carcinogenesis

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutation. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and phase II enzymes that include various transferases. During the past 10 years, considerable attention has been focused on the role of P450s in human cancer susceptibility. Polymorphisms in expression of P450s and transferases exist in humans and these might render increased susceptibility or resistance to cancer. Thus it is important to understanding how P450s participate in the carcinogenesis process and to determine if they are indeed the rate limiting and critical interface between the chemical and its biological activity. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disruption of genes encoding P450s CYP1A2, CYP2E1, and CYP1B1 were developed by use of gene disruption in empbryonic stem cells. Mice lacking expression of microsomal epoxide hydrolase and NADPH:quinone oxidoreductase were also made. These mice exhibit no grossly abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying human carcinogens and validate the molecular epidemiology studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.

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.

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.

Immunohistochemical assessment of human microsomal epoxide hydrolase in primary and secondary liver neoplasm: a quantitative approach.

1. Epoxide hydrolases form an enzyme family involved in the metabolism of a variety of xenobiotics including cytostatic drugs and carcinogens. Whether human microsomal epoxide hydrolase--one of the main members of the epoxide hydrolase family--is expressed in neoplasia of the liver has been the subject of a controversial discussion. 2. We therefore developed a quantitative immunohistochemical assay and monitored epoxide hydrolase expression in hepatocellular carcinomas (HCC, n = 20), cholangio-cellular carcinomas (CCC, n = 2) and liver metastases (n = 57) of tumours of various origins, and compared the expression intensities and patterns to normal liver tissue. 3. In normal liver tissue microsomal epoxide hydrolase displays expression of the constitutive type with non-zonal staining of all hepatocytes. 4. When using a quantitative immunohistochemical approach statistically significant differences in microsomal epoxide hydrolase expression were observed between normal tissue, hepatocellular carcinoma and liver metastases (mean optical density 2.35, 1.63 and 0.21 respectively, p = 2.9, 6.3 and 18.9). These data indicate differential expression in different types of liver neoplasm. 5. As microsomal epoxide hydrolase is involved in metabolism of different xenobiotics our findings may have implications for tumour progression.

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.