Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.     

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.     

Human liver cytosolic epoxide hydrolases

Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHCSO) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7,8-oxide, cis-2-methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1,2-dimethylstyrene oxide and 2,2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2,2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred with trans-1,2-dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes. The isoelectric point for both microsomal styrene 7,8-oxide and cis-stilbene oxide hydrolyzing activity was 7.0, whereas cEHCSO had an isoelectric point of 9.2 and cytosolic trans-stilbene oxide hydrolase (cEHTSO) of 5.7. The cytosolic epoxide hydrolases could be separated by anion-exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHCSO than for cEHTSO. Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents. Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7,8-oxide strongly inhibited cEHCSO whereas cEHTSO was moderately affected by these compounds. Immunochemical investigations revealed a close relationship between cEHCSO and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide. Five steps were necessary to purify cEHCSO. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.     

The major isozyme of rat cardiac glutathione transferases. Its correspondence to hepatic transferase X

A major isozyme of rat heart glutathione transferase was purified to homogeneity by Sephadex G-200 gel filtration, ammonium sulfate precipitation, CM-cellulose chromatography and affinity chromatography on S-hexylglutathione-linked Sepharose 6B. The purified isozyme was a dimer with an apparent relative molecular mass of 50 000 composed of two Yb-size subunits (Mr = 26 500). The isozyme is immunologically related to rat liver glutathione transferase X and 3-3, especially closely to transferase X, and no immunological cross-reactivity with subunits 1 and 2 of hepatic glutathione transferases was observed. The isoelectric point (pI = 6.9) of the isozyme was identical with and the substrate specificity was very similar to transferase X. Thus, the cardiac near-neutral isozyme is considered to be identical to glutathione transferase X recognized in rat liver. The amount of this near-neutral isozyme estimated to be present in heart tissue is 70 micrograms/g. The isozyme has relatively high activities towards alpha, beta-unsaturated carbonyl compounds such as trans-4-phenyl-3-buten-2-one and trans-4-hydroxynon-2-enal. The latter is a cytotoxic product resulting from lipid peroxidation of polyunsaturated fatty acids, and the cardiac isozyme may play a physiologically significant role with glutathione conjugation of this compound. In addition to the near-neutral isozyme, acidic forms with isoelectric points of 4.9, 5.2 and 5.5 were partially purified; some of them are considered to consist of subunits immunologically related to transferase X.     

Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.

The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).     

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Trans-stilbene oxide, trans-β-methylstyrene, 7,8-oxide, trans-β-ethylstyrene, 7,8-oxide, trans-β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his⁻ strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10⁶ of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.     

Cell-contact mediated modulation of the sialylation of contactinhibin

Contactinhibin was found to be involved in contact-dependent inhibition of growth. The growth inhibitory activity of contactinhibin is mediated by N-linked oligosaccharides with desialylated -glycosidically linked, terminal galactose residues. Here we show that in sparse human fibroblasts contactinhibin was expressed in a biologically inactive, highly sialylated form both on the plasma membrane and intracellularily, while in confluent cells plasma membrane localized contactinhibin was present in a biologically active, low sialylated form. Plasma membranes were shown to contain a glycoprotein sialidase which is suggested to be engaged in the activation of contactinhibin in a cell contact-dependent manner.     

Enzymic hydration of benzene oxide: Assay and properties

A radiometric assay for epoxide hydratase using [¹⁴C]benzene oxide as substrate has been developed. The reaction product trans-1,2-[¹⁴C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.     

Relationship between mutagenicity and DNA adduct formation in mammalian cells for fjord- and bay-region diol-epoxides of polycyclic aromatic hydrocarbons

Chinese hamster V79 cells were treated with the anti- and syn-diastereomers of the bay- or fjord-region diol-epoxides of four polycyclic aromatic hydrocarbons, namely benzo[a]pyrene (BP), benzo[c]chrysene (BcC), benzo[g]chrysene (BgC) and benzo[c]phenanthrene (BcPh). The frequency of induction of 6-thioguanine-resistant mutations was determined, and the extent of formation of DNA adducts was measured by 32P-postlabelling. When expressed as mutation frequency per nanomoles compound per millilitre incubation medium, this group of chemicals expressed a 160-fold range in potency. In agreement with previous experimental studies, the anti-diol-epoxide of BcC was highly mutagenic, inducing in excess of 3 x 10(4) mutations/10(6) cells per nmol compound/ml. The mutagenic activities of the anti- and syn-diol-epoxides of BP were 10- and 100-fold lower, respectively. Both diol-epoxides of BgC, the syn-BcC and the anti-BcPh derivatives were also highly mutagenic, and only the syn-BcPh diol-epoxide was less mutagenic than the anti-diol-epoxide of BP. Determination of the levels of DNA adducts formed by the diol-epoxides indicated that the most mutagenic compounds were the most DNA reactive, although the fjord-region diol-epoxides gave rise to more complex patterns of adducts than those of the BP diol-epoxides. When the mutagenicity results were expressed as mutations per femtomoles total adducts formed, all compounds showed similar activities. Thus the potent mutagenicity of the fjord region diol-epoxides appears to be due to the high frequency with which they form DNA adducts in V79 cells, rather than to formation of adducts with greater mutagenic potential.