Presence Of Epoxide Hydrolase Research Articles

Stability of silorane dental monomers in aqueous systems

Siloranes (silicon-based monomers with oxirane functionality) are investigated as matrix resins for new low shrinkage/stress dental composites. Compounds containing oxirane groups are known to be reactive with water, which could impart instability to the composite. To test the stability of siloranes by measuring changes in the chemical structure of the oxirane group in aqueous environments. Two siloranes (PH-SIL and TET-SIL) and their 1:1 mixture (SIL-MIX) were evaluated (n=2-3). Siloranes were mixed in aqueous solutions with and without 1% tetrahydrofuran (THF) containing either liver esterase or epoxide hydrolase at pH 7.4, or dilute HCl at pH 1.4. The stability of conventional dioxiranes 3,4-epoxycyclohexyl-methyl-3,4-epoxycyclohexane carboxylate (ECHM-ECHC), and bisphenol A diglycidyl ether (BADGE) were also monitored under similar conditions. NMR was used to estimate the extent of reaction and give structural information about reaction products. Siloranes were found to be stable for 24h in all aqueous environments tested. In contrast, ECHM-ECHC reacted at pH 1.4 to form species containing oxirane, ester, hydroxyl and carboxylic acid groups. Water hydrolyzed the ester group of ECHM-ECHC in the presence of liver esterase. In the presence of epoxide hydrolase, BADGE oxirane groups were hydrolyzed to diols, hydrolysis ranged from 0 to 34% depending on the aqueous environment. The stability and insolubility of siloranes in biological fluid simulants suggests that these may be more suitable for use in the oral environment than conventional oxirane-functional monomers.

Specificity of 17β-oestradiol and benzo[α]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48Ala119Leu432, Arg48Ala119Val432, Gly48Ala119Leu432, Gly48Ala119Val432, Arg48Ser119Leu432, Arg48Ser119Val432, Gly48Ser119Leu432 and Gly48Ser119Val432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17β-oestradiol and benzo[α]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+·CO versus Fe2+ difference spectra with wavelength maxima at 446nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17β-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17β-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17β-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[α]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17β-oestradiol and benzo[α]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes.

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkylated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.

A quantitative comparison of dibenzo[a,l]pyrene‐DNA adduct formation by recombinant human cytochrome P450 microsomes

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11,12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using 32P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74–82, 1999. Published 1999 Wiley-Liss, Inc.

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11, 12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using (32)P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a, l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74-82, 1999. Published 1999 Wiley-Liss, Inc.

Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes.

The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.

Regio- and stereo-selective metabolism of phenanthrene by twelve cDNA-expressed human, rodent, and rabbit cytochromes P-450.

The regio- and stereoselective metabolism of phenanthrene (PA) by seven cDNA-expressed human and five rodent and rabbit cytochromes P-450 has been examined using reverse-phase and chiral stationary phase high-pressure liquid chromatography (HPLC). Turnover numbers ranged from 0.2 to 55 nmol/min per nmol. Using vaccinia virus expression of P-450 enzymes in Hep G2 cells, m1A1 and m1A2 were found to be the most active P-450s. Of the human P-450s, 1A2 and 2B6 have the highest activity and 2C9 has moderate activity. Using cytochrome P-450s expressed in a lymphoblastoid cell line in presence of epoxide hydrolase (EH), human 1A1 had approximately twice the activity of human 1A2. Regioselectivities for PA metabolism were found to be both isozyme and species-dependent. Stereochemical analysis revealed that the P-450s 1A1, m1A1, m1A2, r2A1, r2B1, PB- and 3MC-treated rat liver microsomes preferentially formed 3R,4R-diol enantiomer (88-97%), whereas rabbit 4B formed the 3S,4S-diol enantiomer (72%). Eleven P-450s, 3MC and PB microsomes preferentially formed 1R,2R-diol enantiomer (80-96%). This is the same stereochemistry as the precursors to some diol epoxides that are potent carcinogens.

The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-dihydrodiol.

The potent carcinogen benzo[a]pyrene (B[a]P) and its metabolite B[a]P trans-7,8-dihydrodiol (7,8-diol) require metabolic activation by the microsomal cytochrome P450s (P450s) to exert several adverse biological effects, including binding to DNA, toxicity, mutagenicity, and carcinogenicity. In the study reported here, we defined the role of each of 12 individual cDNA-expressed cytochrome P450s in the metabolism of B[a]P and 7,8-diol. Human P450s 1A1 and 1A2 were expressed in the absence or presence of epoxide hydrolase (EH) in a human lymphoblastoid cell line, and six human and five rodent and rabbit P450s were expressed from cDNA with vaccinia virus vectors in the hepatoma cell line Hep G2. B[a]P metabolism resulted in nine metabolites (three diols, three quinones, and three phenols), which were separated, identified, and quantitated by high-pressure liquid chromatography. In the human lymphoblastoid cells, human 1A1 metabolized B[a]P at a rate 4.5 times greater than that for 1A2. EH was shown to be directly involved in B[a]P activation, since increasing the amount of EH resulted in less 7-hydroxybenzo[a]pyrene and more 7,8-diol formation. Of the human P450s expressed with the vaccinia virus vectors in Hep G2 cells, 1A2 and 2C9 showed the highest activity and 2B6 showed moderate activity for B[a]P metabolism. Mouse 1A1 had activity 40 times higher than any human, rabbit, or rodent P450s, indicating the potential pitfalls of extrapolating P450 activity across species. Metabolism of the 7,8-diol resulted in six metabolites (four tetrols and two triols). In the lymphoblastoid cells, human 1A1 was shown to be 4.2 times more active than 1A2 for 7,8-diol metabolism. Among human P450s expressed from vaccinia virus, 1A2, 2E1, and 2C9 gave the highest activity, and 2C8 and 3A4 showed moderate activity for 7,8-diol metabolism to the diol epoxides. Again, mouse 1A1 was much more active than any other P450. These studies, in which we determined the capacity of individual P450 in the metabolism and activation of B[a]P and 7,8-diol, may thus lead to a better understanding of how P450s control the detoxification and activation of polycyclic aromatic hydrocarbons.

Presence of epoxide hydrolase and glutathione S-transferase in human pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (PAMs) were obtained from 11 patients by bronchoalveolar lavage. Epoxide hydrolase and glutathione S-transferase in sonicated PAMs were measured using benzo(a)pyrene-4,5-oxide as the substrate. The activity of epoxide hydrolase was 0.24 +/- 0.10 nmol/min/mg protein (mean +/- SD), and of glutathione S-transferase 0.22 +/- 0.12 nmol/min/mg protein. There was a significant difference in enzyme activities in the PAMs from smokers and non-smokers. Epoxides may be metabolized in PAMs.

Benzo[a]pyrene metabolism by purified cytochrome P-450 from 3-methylcholanthrene-treated rats.

The metabolism of benzo[a]pyrene in reconstituted pulmonary mono-oxygenase systems has been studied. Metabolites formed by pulmonary cytochrome P450MC, a major form of pulmonary cytochrome P-450 isolated from 3-methylcholanthrene-treated rats, were analysed by h.p.l.c. The profiles of benzo[a]pyrene metabolites formed by the reconstituted P-450MC systems were different from that obtained with rat-lung microsomes, indicating the presence of several unknown metabolites in the reconstituted systems containing NADPH-cytochrome P-450 reductase and epoxide hydrolase. 3-Hydroxybenzo[a]pyrene was a major product formed by pulmonary cytochrome P-450MC, in the absence or presence of epoxide hydrolase. The addition of purified epoxide hydrolase to the reconstituted systems increased the formation of dihydrodihydroxy-benzo[a]pyrenes, particularly 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene. The 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was the major dihydrodiol formed by pulmonary cytochrome P-450MC. By the addition of epoxide hydrolase the total amount of phenols decreased in parallel with an increased production of dihydrodiol, but the amount of quinones was not changed. Similar results concerning the related production of phenols and dihydrodiols, in the absence and presence of epoxide hydrolase, were obtained in reconstituted systems of hepatic cytochrome P-450MC, the major form of hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.

Induction of cytochrome P-450-dependentmonooxygenase systems in embryos and eleutheroembryos of the killfish Fundulus Heteroclitus

Microsomal aryl hydrocarbon hydroxylase (AHH) activity was highly inducible by polychlorinated biphenyls (PCBs) in Fundulus embryos, and stages prior to the appearance of the liver rudiment were competent to respond to these inducers. Consistent with previous observations, basal AHH activity in whole eleutheroembryo microsomes was shown to increase about 9-fold within 24 h of hatching. Aminopyrine N-demethylase (APD) activity also increased with time after hatching. However, the increase in APD activity was much less than that of AHH activity, suggesting a post-hatching change in basal cytochrome P-450 isozyme composition. Also associated with hatching was an increase in the sensitivity to PCBs as inducers of AHH activity. The ED50 for induction of AHH activity in eleutheroembryos was estimated to be only one-third to one-fourth that in embryos. This developmental increase in the sensitivity to PCBs was not due to a redistribution of PCBs between the yolk and tissues with yolk absorption, and was not simply age-dependent, but appeared to require hatching. An additional change in the monooxygenase system associated with hatching was that microsomal NADPH-cytochrome c reductase activity was not inducible by PCBs prior to hatching, but was modestly inducible after hatching. High performance liquid chromatographic (HPLC) analysis of benzo[a]-pyrene (BP) metabolites formed by microsomes from control and PCB-treated eleutheroembryos demonstrated production of dihydrodiols in the 7,8- and 9,10-positions of the benzo-ring. The formation of these metabolites was completely inhibited by the epoxide hydrolase (EH) inhibitor, trichloropropene oxide, indicating the presence of EH in Fundulus eleutheroembryos. Furthermore, these results indicate the Fundulus eleutheroembryos probably can activate BP to its ultimate carcinogenic form, the 7,8-dihydrodiol-9,10-epoxide, and induction of AHH activity by PCBs is likely to increase the rate of formation of activated metabolites from BP and related compounds. However, during the most active period of organogenesis, prior to hatching, basal AHH activity was low, and prehatching stages were relatively insensitive to cytochrome P-450 inducers. The combination of these effects may help to protect these stages from damage from activated metabolites.

Presence of benzo[a]pyrene metabolizing activities in isolated rat liver nucleoli

A benzo[a]pyrene (B[a]P) hydroxylase activity and epoxide hydrolase (EH) activity have been found in rat liver nucleoli obtained from untreated (C) and 3-methylcholanthrene (3-MC) pretreated rats. A comparison of the enzyme activities was made in rat nuclei and nucleoli. Light and electron microscopic observations of nucleolar preparations did not reveal significant contamination either by intact nuclei or by nuclear membranes, and glucose 6-phosphatase, a marker of microsomal activity, was not detected in our nucleolar preparations. NADPH cytochrome c-reductase could be measured in C and 3-MC nuclei and very low but detectable activity was found in the nucleoli. Nucleolar preparations revealed significant hydroxylating activity, which was inducible by 3-MC in nuclei but not in nucleoli. The presence of EH in nucleoli was demonstrated with phenanthrene 9,10-oxide and B[a]P 4,5-oxide, but the nucleolar activities were lower than those obtained using intact nuclei. The addition of 1,1,1-trichloropropene 2,3-oxide completely inhibited EH activity. Furthermore the presence of covalently-bound metabolites of B[a]P formed in isolated nucleoli was demonstrated by cytofluoromicroscopy.