0000000000276603
AUTHOR
Heinz Frank
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…
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.
Abstract 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 10 6 of the activity of the positive control, benzo[ a ]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans -β-methy…
Formation of N-methylnicotinamide in the brain from a dihydropyridine-type prodrug
The enhancement of brain choline levels is a possible therapeutic option in neurodegenerative diseases; however, brain choline levels are held within narrow limits by homeostatic mechanisms including the rapid clearance of excess choline from the brain. The present study tests whether N-methylnicotinamide (NMN), an inhibitor of the outward transport of choline from the brain, can elevate brain choline levels in vivo. As NMN does not cross the blood-brain barrier, we synthesized and administered the brain-permeable prodrug, 1,4-dihydro-N-methyl-nicotinamide (DNMN), and tested its effect on the levels of NMN and choline in brain extracellular fluid, using the microdialysis procedure. Administ…
Sulfotransferase-mediated mutagenicity of 1-hydroxymethylpyrene and 4H-cyclopenta[def]chrysen-4-ol and its enhancement by chloride anions.
1-Hydroxymethylpyrene (HMP), a primary benzylic alcohol, and 4H-cyclopenta[def]chrysen-4-ol (OH-CPC), a secondary benzylic alcohol, were investigated for mutagenicity in Salmonella typhimurium (reversion of the his- strain TA98) in the presence of various xenobiotic-metabolizing systems. In the direct test, HMP was inactive and OH-CPC was very weakly active. In the presence of NADPH-fortified postmitochondrial fraction from rat liver (S9/NADPH), no activation of OH-CPC was observed, whereas strong mutagenic effects were elicited by HMP. In the presence of cytosol and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), both alcohols were activated to potent mutagens. For equal mutagenic effects, a…
Quinone reduction and redox cycling catalysed by purified rat liver dihydrodiol/3 alpha-hydroxysteroid dehydrogenase.
A highly active preparation of rat liver dihydrodiol/3 alpha-hydroxysteroid dehydrogenase was obtained using a newly developed, rapid purification scheme involving affinity chromatography on Red Sepharose. Depending on the coenzyme present, the purified enzyme was found to catalyse the oxidation of dihydrodiols and steroids or the reduction of substrates with carbonyl or quinone moieties. Using a wide range of synthetic quinones derived from polycyclic aromatic hydrocarbons (PAHs), we observed a pronounced regioselectivity of the quinone reductase activity. Good substrates were the o-quinones of phenanthrene, benz(a)anthracene, chrysene and benzo(a)pyrene with the quinonoid moiety in the K-…
Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies
Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mA…
Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates
Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude micros…
Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase
Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the e…
Detection of Reactive Quinones in the Metabolism of Polycyclic Aromatic Hydrocarbons by the Formation of their Glutathione Conjugates
Abstract The biotransformation of polycyclic aromatic hydrocarbons to quinones by rat liver microsomes was investigated. The employment of an electrochemical detector allowed the specific detection of quinones separated by reverse phase HPLC with higher sensitivity as compared to UV detection. Microsomal incubations of benzo[a]pyrene (BP) resulted in the formation of 1,6-, 3,6- and 6,12-quinones, of naphthalene in the detection of naphthalene-1,4-quinone, whereas ortho-quinones could only be detected in trace amounts. Additional protein binding studies showed that only 9–22% of synthetic ortho-quinones could be recovered from microsomal incubations. In order to scavenge possible reactive qu…
Influence of the level of cytosolic epoxide hydrolase on the induction of sister chromatid exchanges by trans-beta-ethylstyrene 7,8-oxide in human lymphocytes.
Abstract trans -β-Ethylstyrene 7,8-oxide, a substrate of cytosolic epoxide hydrolase, and 4-fluorochalcone oxide, an inhibitor of this enzyme, were investigated on induction of sister chromatid exchanges (SCE) in human lymphocytes. Both epoxides enhanced the frequency of SCE. 4-Fluorochalcone oxide at low concentration (2.5μM) inhibited cytosolic epoxide hydrolase activity towards trans -β-ethylstyrene 7,8-oxide in lymphocytes by 74% and had no effect on glutathione transferase activity using this substrate. At this concentration it did not induce SCE itself, but it potentiated the effect of trans -β-ethylstyrene 7,8-oxide several fold. In lymphocytes from different subjects, the number of …
DNA adduct levels associated with p53 induction and delay of MCF-7 cells in S phase after exposure to benzo[g]chrysene dihydrodiol epoxide enantiomers.
Optically active isomers of a mammary carcinogen, anti-benzo[g]chrysene 11, 12-dihydrodiol 13, 14-epoxide, react to different extents with DNA and generate DNA adducts that differ in their stereochemistry. In the study reported here, the effect of these two enantiomers on the progress of human breast carcinoma MCF-7 cells through the cell cycle was investigated. Each enantiomer caused the cells to accumulate in the S phase, but a higher dose of the benzo[g]chrysene 11S, 12R-dihydrodiol 13R, 14S-epoxide than of its enantiomer was required to induce this effect. Similarly, induction of p53 also required a higher dose of benzo[g]chrysene 11S, 12R-dihydrodiol 13R, 14S-epoxide. Postlabeling stud…
Synthesis, Absolute Configuration, and Bacterial Mutagenicity of the 8 Stereoisomeric Vicinal Diol Epoxides at the Terminal Benzo Ring of Carcinogenic Dibenz[a,h]anthracene
The synthesis of the 8 possible stereoisomeric diol epoxides (DEs) at the terminal benzo ring of carcinogenic dibenz[a,h]anthracene (DBA) is reported. trans-3,4-Dihydroxy-3,4-dihydro-DBA (1) afforded the 4 bay region DEs: the enantiomeric pairs of the anti diastereomers (+)-3/(-)-3 and of the syn diastereomers (-)-4/(+)-4, respectively. trans-1,2-Dihydroxy-1,2-dihydro-DBA (2) served as precursor of the 4 reverse DEs: the enantiomeric pairs of the anti diastereomers (+)-5/(-)-5 and of the syn diastereomers (-)-6/(+)-6, respectively. The transformation of the olefinic double bond in the enantiomeric trans-dihydrodiols to epoxides was achieved by either (i) oxidation with m-chloroperoxybenzoic…
Regio- and stereoselectivity in the metabolism of benzo[c]phenanthrene mediated by genetically engineered V79 Chinese hamster cells expressing rat and human cytochromes P450.
Regio- and stereoselective metabolism mediated by cytochrome P450 (CYP) and metabolite-dependent cytotoxicity of benzo[c]phenanthrene (B[c]Ph) and its trans-3,4-dihydrodiol, the metabolic precursor of the carcinogenic fjord-region B[c]Ph-3,4-dihydrodiol 1,2-epoxides (B[c]PhDE), were investigated with V79 Chinese hamster cells genetically engineered for three rat and six human CYP isoforms. The order of the capabilities of the CYP isoforms to metabolize B[c]Ph was as follows: h1A1>r1A1>r1A2>h1B1>h1A2>r2B1>>h2E1>h2A6>h3A4. Regardless of the species, all individual CYP isoforms preferentially catalyzed the oxidation of B[c]Ph at the 5,6-position (K-region) except human CYP1A1 and human CYP1A2,…
Microsomal activation of dibenzo[def,mno]chrysene (anthanthrene), a hexacyclic aromatic hydrocarbon without a bay-region, to mutagenic metabolites.
Metabolically formed dihydrodiol epoxides in the bay-region of polycyclic aromatic hydrocarbons are thought to be responsible for the genotoxic properties of these environmental pollutants. The hexacyclic aromatic hydrocarbon dibenzo[def,mno]chrysene (anthanthrene), although lacking this structural feature, was found to exhibit considerable bacterial mutagenicity in histidine-dependent strains TA97, TA98, TA100, and TA104 of S. typhimurium in the range of 18-40 his(+)-revertant colonies/nmol after metabolic activation with the hepatic postmitochondrial fraction of Sprague-Dawley rats treated with Aroclor 1254. This mutagenic effect amounted to 44-84% of the values determined with benzo[a]py…
Direct optical resolution of trans-dihydrodiol enantiomers of fjord-region polycyclic aromatic hydrocarbons by high-performance liquid chromatography on a modified cellulose phase
Abstract Enantioselective separation of trans -dihydrodiol metabolites of a series of fjord-region polycyclic aromatic hydrocarbons (PAHs), such as benzo[ c ]phenanthrene and dibenzo[ a , l ]pyrene, was evaluated by HPLC using commercially available cellulose-based CSPs as chiral columns. A baseline separation ( R s ≥1.6) with sharp, well-defined peaks of individual enantiomers was attained using cellulose-tris-( N -3,5-dimethylphenylcarbamate) and n -heptane-ethanol (9:1, v/v) as mobile phase. These chromatographic conditions permit a direct, simple and rapid (mostly within 30 min) enantiomeric resolution of PAH dihydrodiols. CD spectra were obtained for all optically pure enantiomers and …
Role of the Ha-ras gene in the malignant transformation of rat liver oval cells.
We have shown that the oval cell line OCICDE 22 can be transformed by the highly carcinogenic fiord-region diol epoxides of benzo[c]phenanthrene. Mutational activation of the ras proto-oncogene family has been proposed to be a critical event in the formation of tumors induced by polycyclic aromatic hydrocarbons. Therefore, we investigated whether in the earlier transformed OCICDE 22 cells any point mutations were detected in the ras proto-oncogene. The results indicate that the malignant transformation of OCICDE 22 cells by the 4 stereoisomeric benzo[c]phenan-threne diol epoxides in vitro is independent of activation of the Ha-ras proto-oncogene. In addition, Northern and Western blot analy…
Stereoselective metabolism of dibenz(a,h)anthracene to trans-dihydrodiols and their activation to bacterial mutagens.
Dibenz(a,h)anthracene (DBA), a carcinogenic, polycyclic aromatic hydrocarbon ubiquitous in the environment, is metabolized by the hepatic microsomal fraction of immature Sprague-Dawley rats pretreated with Aroclor 1254 to 27 ethyl acetate-extractable metabolites. More than half of these metabolites (51%) consisted of trans-1,2-; -3,4-; and -5,6-dihydrodiols including their identified secondary metabolites. The three trans-dihydrodiols (4.9, 15.8, and 0.6% of total metabolic conversion) were highly enriched in their R,R enantiomers (85, 71, and 98%) as determined by high performance liquid chromatography on suitable chiral stationary phases. This is explained on the basis of the stereoselect…