Studies on the importance of microsomal epoxide hydrolase in the detoxification of arene oxides using the heterologous expression of the enzyme in mammalian cells.

In order to investigate the role of the microsomal epoxide hydrolase (mEH) in the detoxification of arene oxides in the presence of a high endogenous glutathione S-transferase (GST) activity-a situation found in several organs--we expressed the rat mEH cDNA in BHK21 Syrian hamster cells. These cells have high GST activities but contain an extremely low endogenous mEH enzyme activity. We obtained several cell clones which expressed the mEH heterologously, as determined by immunoblotting. The cell clone BHK21-mEH/Mz1 had the highest level of mEH protein. Immunofluorescence showed that the level of expression was almost homogeneous throughout the cell population. Total protein isolated from th…

Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis

Direct comparison of the amino acid sequences of microsomal and soluble epoxide hydrolase superficially indicates that these enzymes are unrelated. Both proteins, however, share significant sequence similarity to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the alpha/beta hydrolase fold family of enzymes. The catalytic mechanism for the dehalogenase has been elucidated in detail [Verschueren et al. (1993) Nature 363, 693-698] and proceeds via an ester intermediate where the substrate is covalently bound to the enzyme. From these observations we conclude (i) that microsomal and soluble epoxide hydrolase are distantly related enzymes that have evolved from a co…

Toxicological implications of enzymatic control of reactive metabolites.

Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human req…

Dynamics of the localization of drug metabolizing enzymes in tissues and cells.

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamste…

Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes

We present data showing that the major phenobarbital inducible cytochromes P-450 (cytochrome P-450IIB1 and cytochrome P-450IIB2) were phosphorylated in intact hepatocytes. This phosphorylation was greatly increased by the cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP mediated by a cAMP-dependent protein kinase. Most importantly the phosphorylation status of cytochromes P-450 was shown to change in the hepatocytes after treatment with glucagon, which is known to increase the level of cAMP in hepatocytes. The observed impact of the hormone glucagon on the phosphorylation of distinct cytochrome P-450 forms in intact hepatocytes reveals the possibility that the enzyme activity of cyt…

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…

Differential Effects of Fluvoxamine and Other Antidepressants on the Biotransformation of Melatonin

Melatonin, the predominant product of the pineal gland, is involved in the maintenance of diurnal rhythms. Nocturnal blood concentrations of melatonin have been shown to be enhanced by fluvoxamine, but not by other serotonin reuptake inhibitors. Because fluvoxamine is an inhibitor of several cytochrome P450 (CYP) enzymes, the authors studied the biotransformation of melatonin and the effects of fluvoxamine on the metabolism of melatonin in vitro using human liver microsomes and recombinant human CYP isoenzymes. Melatonin was found to be almost exclusively metabolized by CYP1A2 to 6-hydroxymelatonin and N-acetylserotonin with a minimal contribution of CYP2C19. Both reactions were potently in…

Evidence for Several Hepatic Proteins Related to Microsomal Epoxide Hydrolase

Epoxide hydrolases catalyze the conversion of epoxides, some of which have been shown to be carcinogenic, to dihydrodiols (Guenthner and Oesch 1981). At least three forms of epoxide hydrolases exist in rats, two of which, namely mEHb and mEHch, are associated mainly with the microsomal fraction (Oesch et al 1984; Levin et al 1983) whereas one form namely cEH is found to a large extent in the cytosolic fraction (Gill and Hammock 1981). These three forms differ in their immunological and catalytic properties quite considerably (Guenthner et al 1981). In the case of mEHb the existence of several closely related isoenzymes with an identical apparent subunit molecular weight (Mrs) of 50,000 was …

DNA Polymerase Action on Oligonucleotide Templates from Human Ha-rasProtooncogene Containing N6-Deoxyadenosine Adducts Derived from Trans Addition of (+)- and (-)-anti-Benzo[c]phenanthrene-3,4-dihydrodiol 1,2-epoxides at Codon 61

Abstract In the present work we have used a DNA polymerase assay to investigate the primer extension with T7 DNA polymerase (Sequenase 2.0) and the Klenow fragment of Escherichia coli DNA polymerase I (exo − KF) on chemically synthesized 21mer templates representing partial sequences of the human Ha-ras protooncogene with site-specifically positioned trans-N 6-dA adducts of (-)- (adduct 1) and (+)-anti-benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides (adduct 2) at codon 61 (CA∗G; A∗ indicates the adducted position). With Sequenase 2.0 a complete block of primer extension opposite both adduct 1 and 2 was noted using a 10mer primer reaching the (n-1)-position of the adduct. A detailed analys…

The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in whic…

Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins

A fast and sensitive procedure for the colorimetric detection of sodium lauryl sulfate (SDS) is presented. The assay is based upon the formation of a chloroform-extractable ion pair between lauryl sulfate and methylene blue that is quantified spectrophotometrically with an estimated detection limit of 150 ng of SDS. The method is suitable for the monitoring of contaminating traces of SDS in protein or nucleic acid samples that have the potential to interfere with enzymatic manipulations such as proteolytic digest, restriction analysis, or reverse transcription. Since the procedure is extremely simple and no special equipment is required it is accessible to every researcher concerned with SD…

Mechanism-based predictions of interactions.

Abstract Exposure to more than one toxic compound is common in real life. The resulting toxic effects are often more than the simple sum of the effects of the individual compounds. It is unlikely that it will ever be possible to test all combinations. It is therefore highly desirable to improve or develop means for reasonably approximating predictions of interactions. In order to be valid and extrapolatable, these predictions are most promising if they are mechanism-based. Examples will be given for possibilities of mechanism-based predictions of interactions which exceed trivialities of simple increases by enzyme induction of enzymatic rates of a given biotransformation pathway leading to …

Sequence of a novel cytochrome CYP2B cDNA coding for a protein which is expressed in a sebaceous gland, but not in the liver

The major phenobarbital-inducible rat hepatic cytochromes P-450, CYP2B1 and CYP2B2, are the paradigmatic members of a cytochrome P-450 gene subfamily that contains at least seven additional members. Specific oligonucleotide probes for these genomic members of the CYP2B subfamily were used to assess their tissue-specific expression. In Northern-blot analysis a probe specific to gene 4 (which is designated now as CYP2B12) hybridized to a single mRNA present in the preputial gland, an organ which is used as a model for sebaceous glands, but did not hybridize to mRNA isolated from the liver or from five other tissues of untreated or Aroclor 1254-treated rats. The cDNA sequence for the CYP2B12 R…

Stable Expression of Heterologous Microsomal Epoxide Hydrolase in BHK21 Cells: Influence on the Mutagenicity of Benzo[a]pyrene 4,5-Oxide

Most environmental mutagens and carcinogens require metabolic activation to electro- philic intermediates capable of reacting with cellular target structures, such as DNA. These electrophilic intermediates are in addition subject to metabolic detoxification. This metabolism is mainly controlled by enzymes whose expression is very variable. Among other things, various enzymes are inducible by environmental chemicals. Understanding the toxicology of chemicals (for example, species differences, idiosyncrasias, organotropisms) therefore requires knowledge of critical host factors. One approach towards this goal involves the use of purified enzymes in metabolism and toxicological studies (Glatt …

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produc…

Selective detection of mRNA forms encoding the major phenobarbital inducible cytochromes P450 and other members of the P450IIB family by the RNAse A protection assay.

Abstract The identification of P450 mRNAs in a tissue poses the problem that members of the same P450 gene family share a high sequence homology. Studies based on oligomer probes rely on a probe covering only a few base pairs. In contrast in our study on the expression of the P450IIB gene family we used in vitro-generated antisense transcripts, covering several hundred base pairs, of the hypervariable and constant regions of the P450IIB1 and P450IIB2 cDNA, in the RNAse A protection assay of mRNA isolated from various tissues. RNAse A concentrations were adjusted to a level where this enzyme still yielded distinct fragments for a defined P450IIB1 antisense/P450IIB2 sense heteroduplex, which …

Introduction of Cytochrome P-450 Genes into V79 Chinese Hamster Cells to Generate New Mutagenicity Test Systems

Usually, cultivated cells have poor capabilities to metabolize promutagens and procarcinogens. This is particularly true for cells that grow fast and have a high cloning efficiency, as is the case with V79 Chinese hamster cells. For this reason, these cells are being extensively used in mutagenicity tests. But, due to the fact that particularly these cells lack cytochrome P-450 activities, promutagens and procarcinogens have to be incubated with an exogenous metabolizing system, e.g. liver homogenate preparations, in order to generate reactive metabolites. These extracellularly generated metabolites are then given to V79 cells in order to check for their potency to mutate the chromosomal DN…

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes.

Abstract A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to devide into isoenzymes phosphorylated (i) by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), (ii) by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and (iii) by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor subs…

Significance of Posttranslational Modification of Drug Metabolizing Enzymes by Phosphorylation for the Control of Carcinogenic Metabolites

The total activity of foreign compound metabolizing enzymes may change by altering the amount or the specific activity of the enzyme by induction or repression, or by activation or inhibition. The important contribution of enzyme induction is well known (Conney 1982, Oesch 1986, Nebert and Jones 1989). This is a relatively slow process which requires the biosynthesis of the enzyme protein. The possibility of a faster regulation of foreign compound metabolism by posttranslational modification by phosphorylation of an already preexisting protein molecule has only recently received attention. A central role in the metabolism of foreign compounds is played by the cytochrome P450-dependent monoo…

Investigating the Role of the Microsomal Epoxide Hydrolase Membrane Topology and Its Implication for Drug Metabolism Pathways

The microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of reactive epoxides which are formed by the action of cytochromes P450 from xenobiotics. In addition the mEH has been found to mediate the transport of bile acids. For the mEH it has been shown that it is cotranslationally inserted into the endoplasmic reticulum. Here we demonstrate that the amino-terminal twenty amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can be also supplied by a cytochrome P450 (CYP2B1) anchor signal sequence.

Studies of the expression of the cytochrome P450IA, P450IIB, and P450IIC gene family in extrahepatic and hepatic tissues.

We have studied the expression of three P-450 gene subfamilies in hepatic and extrahepatic tissues using the sensitive RNAse A protection assay. Members of the P450IA subfamily, which encodes the major methylcholanthrene-inducible cytochromes P-450, were found to be not expressed in extrahepatic tissues of untreated animals, raising the question whether these P-450 play a role in the metabolism of carcinogens in unexposed individuals. In contrast, members of the P450IIB family, some of which encode the major phenobarbital-inducible cytochromes P-450, were found to be expressed in some extrahepatic tissues of untreated rats and here most notably in the lung and in sebaceous glands. Members o…

The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology

Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been …

C-erbB-2-oncogene expression in breast carcinoma: Analysis by S1 nuclease protection assay and immunohistochemistry in relation to clinical parameters

The c-erbB-2 mRNA was detected by the S1 nuclease protection assay and Northern blotting in breast cancer tissues. In contrast to the Northern blot analysis which has been used in all recent publications concerning c-erbB-2 expression on the level of RNA, the S1-nuclease protection assay has distinct advantages with respect to sensitivity, reproducibility, and handling of radioactive probes. We compared the expression of c-erbB-2 in 120 breast carcinomas which were operated in the years 1989-1990 on the level of the mRNA (S1 nuclease protection assay) and the protein (immunohistochemistry), respectively. In general, results obtained with both methods were in good agreement. Only minor diffe…