Reaktive Metabolite cancerogener polycyclischer Kohlenwasserstoffe: Synthese und Abfangreaktion von 9-Hydroxybenzo[a]pyren-4,5-oxid

Le compose du titre est prepare a partir de l'acetate-9 de benzo [a] pyrene et piege par l'ethanethiol. Mecanismes

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

Abstract 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, inducin…

Formation of mono- and diglucuronides and other glycosides of benzo(a)pyrene-3,6-quinol by V79 cell-expressed human phenol UDP-glucuronosyltransferases of the UGT1 gene complex

Glucuronidation of quinols of polycyclic aromatic hydrocarbons (PAHs) represents an important detoxication pathway preventing toxic quinone/quinol redox cycles. Therefore, mono- and diglucuronide formation of benzo(a)pyrene-3,6-quinol was investigated and compared to that of structurally related 3,6-dihydroxychrysene and simple phenols (1-naphthol and 4-methylumbelliferone) using V79 cell-expressed human UGT1.6 (= P1) and human UGT1.7 (= P4). Properties of human UGT1.6 were compared to those of the rat ortholog. Cofactors related to UDP-glucuronic acid such as UDP-galacturonic acid and UDP-glucose were also studied. It was found that rat and human UGT1.6 and human UGT1.7 catalyse monoglucur…

Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol) was isolated from arylsulfatase/beta-glucuronidase-treated bile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) has been administered. This triol was investigated for mutagenicity in Salmonella typhimurium (reversion to histidine prototrophy of strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine). When no exogenous metabolizing system was added the triol was inactive, while 3-OH-BP showed weak mutagenic effects with all four bacterial strains. In the presence of NADPH-fortified postmitochondrial supernatant fraction (S9 mix) of liver homogenate fro…

DNA Modification Induced After Metabolic Activation of the Potent Carcinogen Dibenzo[a, l]pyrene in V79 Chinese Hamster Cells Stably Expressing Single Cytochromes P450

Abstract The polycyclic aromatic hydrocarbon (PAH) dibenzo[a, l]pyrene (DB[a, l]P) has been found to be an environmental pollutant and, considering the available data from rodent bioassays, it represents the most carcinogenic member compound of the class of PAH yet discovered. To sort out the contribution of individual cytochromes P450 (P450) in the metabolic activation of this PAH, V79 cells stably expressing a single P450 isoform were treated with DB[a, l]P or enantiomeric DB[a, l]P-11,12-dihydrodiols (diols). Subsequent analysis of the DNA adducts formed revealed substantial differences in the adduct pattern and the total DNA binding depending on the cell line used. Human P450 1B1 effect…

Synthesis and Characterization of Adducts Derived from the syn-Diastereomer of Benzo[a]pyrene 7,8-Dihydrodiol 9,10-Epoxide and the 5‘-d(CCTATAGATATCC) Oligonucleotide

5'-d(CCTATAGATATCC) was reacted with each syn-enantiomer of trans-7,8-dihydroxy 9,10-epoxy 7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE). The (-)-enantiomer yielded one dominating adduct, whereas the (+)-enantiomer resulted in two major adducts. As indicated by optical spectroscopic methods, the major adduct derived from both (-)- and (+)-syn-BPDE involves cis addition of the C-10 position of the diol epoxide to the exocyclic amino group of deoxyguanosine [(-)-syn-BPDEc-N2-dG and (+)-syn-BPDEc-N2-dG, respectively], whereas the minor (+)-syn-BPDE adduct is identical to a trans adduct [(+)-syn-BPDEt-N2-dG]. The cis adducts as well as the (+)-syn-BPDEt-N2-dG adduct are chemically stable for sev…

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene

A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmo…

Mono- and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7

Polycyclic aromatic hydrocarbon (PAH)-type compounds induce at least two rat UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT1A7. Among the glucuronidation reactions of PAH metabolites studied, mono- and diglucuronide formation of benzo[a]pyrene and chrysene-3,6-diphenol showed the highest induction factors in rat liver microsomes. Availability of AHH-1 cells stably expressing UGT1A7 allowed us to study whether this PAH-inducible isoform could catalyze benzo[a]pyrene and chrysene-3,6-diphenol glucuronidation. It was found that UGT1A7 indeed catalyzed mono- and diglucuronide formation of both benzo[a]pyrene and chrysene 3,6-diphenols. V79 cell-expressed rat UGT1A6 also catalyzed these re…

A facile microsynthesis of 14C-labelled picene

Microscale Wittig olefination of commercially available [7-14C]-benzal dehyde of high specific radioactivity with aryl bromomethyl phosphonium salt and subsequent oxidative photocyclisation affords pure 14C-labelled picene in efficient yield.

Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems

Abstract Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules leading to electrophilic cations which may form adducts with DNA and other important cellular structures. Since endogenous sulfotransferases do not appear to be expressed in indicator cells of standard mutagenicity tests, rat and human sulfotransferases have been stably expressed in his−Salmonella typhimurium strain TA1538 and Chinese hamster V79 cells. Using these recombinant indicator cells, sulfotransferase-dependent genotoxic activities were detected…

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…

Metabolic activation of dibenzo[a,l]pyrene by human cytochrome P450 1A1 and P450 1B1 expressed in V79 Chinese hamster cells.

Metabolic activation of the strongly carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) and its trans-8,9-dihydrodiol (trans-8,9-diol) catalyzed by human cytochromes P450 (P450) 1A1 and 1B1 was investigated. DNA binding of DB[a,l]P in mammalian cell lines has previously been shown to be preferentially mediated by fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE). In order to elucidate different capabilities of both P450 enzymes for metabolic activation of DB[a, l]P V79 Chinese hamster cells, stably expressing human P450s 1A1 or 1B1 have been exposed to the parent PAH or its racemic trans-8, 9-diol. For this purpose, synthesis and spectroscopic…

Conjugation reactions of polyaromatic quinones to mono- and bisglutathionyl adducts: Direct analysis by fast atom bombardment mass spectrometry

The conjugation products of several reactive quinones with glutathione have been identified by fast atom bombardment mass spectrometry. Appropriate conditions have been developed which enabled the direct, dynamic mass spectral analysis of spontaneous, as well as glutathione transferase catalysed conjugation reactions. Applications to a series of quinones provided the direct detection and differentiation of the formation of mono- and bisglutathionyl adducts between regioisomeric quinone substrates in that only 1,4-quinones yielded bisglutathionyl adducts, which were not observed for the 1,2-isomers.

Activating mutations in human c-Ha-ras-1 protooncogene induced by stereoisomeric fjord-region benzo[c]chrysene diol-epoxides.

The mutagenicity of fjord-region benzo[c]chrysene diol-epoxide (BcCDE) stereoisomers((+) anti-BcCDE, (-)anti-BcCDE, (+)syn-BcCDE, and (-)syn-BcCDE) was studied in a forward-mutation system. pEC plasmid containing the human c-Ha-ras-1 proto-oncogene was reacted in vitro with each optically active isomer separately and transfected into NIH/3T3 cells. Morphologically transformed foci were cloned, and DNA obtained from these foci was tested for the presence of Ha-ras-1 sequence by Southern blot analysis. A total of 50 transformed foci (11-14 for each diastereomer) were generated. To determine the nature of mutations responsible for activating the proto-oncogene, regions of the gene likely to co…

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…

Chiral Inversion of 1-Hydroxyethylpyrene Enantiomers Mediated by Enantioselective Sulfotransferases

The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases. The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol. Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer. The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an SN1 mechanism and therefore produces racemic alcohol. hEST showed high enantioselectivity for S-1-HEP, whereas hHST strongly preferred the R-enantiomer. The r…

Spectroscopic Studies of Oligonucleotide Adducts and Base Sequence Preference of Adducts Formed by the Stereoisomers of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-Tetrahydrobenzo[a]pyrene

Abstract 5′-d(CCTATAGATATCC) has been reacted with BPDE and the adducts derived from binding of BPDE to the exocyclic amino group of deoxyguanosine (dG) were studied with spectroscopic methods. The major dG-adducts of (+)- and (-)-anti-BPDE and a minor adduct of (+)-syn-BPDE showed the characteristics of trans-adducts. The major products formed with (+)- and (-)-syn-BPDE exhibit cis-adduct characteristics. Annealing of BPDE-modified oligonucleotides to complementary or partially complementary strands results in reduced fluorescence intensity in several cases and in others the intensity is markedly increased. These differences demonstrate that the adduct microenvironment is strongly influenc…

Metabolism of Phenanthrene, Benz[a]anthracene, Benzo[a]pyrene, Chrysene and Benzo[c]phenanthrene by Eight cDNA-expressed Human and Rat Cytochromes P450

Abstract Phenanthrene, benz[a]anthracene, chrysene, benzo[c]phenanthrene, and benzo[a]pyrene have been studied for their regiospecific oxidation by five human (1A1, 1A2, 2A6, 2E1, 3A4) and three rat (1A1, 1A2, 2B1) CYP isoforms. All substrates are preferentially metabolized by CYP1A1 and CYP1A2 in human and rat. Other isoforms play a minor role if at all. Significant differences between human and rat CYP isoforms can be recognized with regard to the regiospecific oxidation of PAH. For instance, K-region oxidation is more pronounced in rat than in human CYP1A1 and CYP1A2. Hence, extrapolation from metabolism studies in rodents to human may be limited.

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…

Enhancement of the Mutagenicity of Ethylene Oxide and Several Directly Acting Mutagens by Human Erythrocytes and its Reduction by Xenobiotic Interaction

According to the present state of knowledge mutagenicity or genotoxicity of the ulti mate genotoxic agents ethylene oxide or styrene oxide cannot be increased by further me tabolism. However, in the present study we demonstrate that mutagenicity of several ultimate genotoxic substances is increased by human erythrocytes. For instance mu tagenicity of mafosfamide, N-nitroso-N-methylurea, ethylene oxide, and styrene oxide to Salmonella typhimurium TA 1535 was increased 5.5-, 5.1-, 2.7-, and 2.3-fold, respectively, by addition of human erythrocyte homogenate to the preincubation mixture in the Ames test. On the other hand, the mutagenicity of cumene hydroperoxide, benzo[a]pyrene-4,5-oxide, and…

Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo[a,l]pyrene.

Extensive tumorigenicity studies in rodents revealed that dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen among all polycyclic aromatic hydrocarbons (PAHs) tested so far. The structure of the genotoxic metabolite(s) responsible for this exceptional carcinogenicity is unknown. The fjord-region syn- and anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (syn- and anti-DB[a,l]PDE) were synthesized to clarify their role as possible ultimate mutagenic and carcinogenic metabolites of DB[a,l]P.9-Formyl-11,12-dimethoxybenzo[g] chrysene was prepared from 9-phenanthrylacetic acid by a photochemical route. After reaction of the aldehyde with trimethylsulfonium iodide to generate an oxiranyl si…

Applications of stable V79-derived cell lines expressing rat cytochromes P4501A1, 1A2, and 2B1.

1. Chinese hamster V79-derived cell lines, stably expressing cytochromes P4501A1, 1A2, and 2B1 activities, were constructed by genetic engineering in continuation of our work to establish a battery of V79 derived cell lines designed to study the metabolism of xenobiotics. 2. Cell lines XEM1 and XEM2, expressing cytochrome P4501A1, were capable of the O-dealkylation of 7-ethoxycoumarin and the hydroxylation of benzo[a]pyrene. 3. Cell lines XEMd.MZ and XEMd.NH, expressing P4501A2, were shown to hydroxylate 17 beta-estradiol and 2-aminofluorene. 4. Cell line SD1, expressing cytochrome P4502B1, was able to hydroxylate testosterone stereo- and regio-specifically at the 16 alpha and 16 beta posit…

The cytotoxicity of mitomycin C and Adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytot…

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…

Tumor-initiating activity of the (+)-(S,S)- and (−)-(R,R)-enantiomers of trans-11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene in mouse skin

Abstract A single administration of enantiomerically pure 11,12-dihydrodiols of dibenzo[ a,l ]pyrene (DB[ a,l ]P) on the back of NMRI mice and subsequent chronic treatment with 12- O -tetradecanoylphorbol 13-acetate (TPA) (initiation/promotion assay) revealed strikingly different carcinogenic activities of both enantiomers. Tumor-initiating activity of (−)-(11 R ,12 R )-DB[ a,l ]P-dihydrodiol, which is the metabolic precursor of the (−)- anti -(11 R ,12 S )-dihydrodiol (13 S ,14 R )-epoxide, was exceptionally higher than the corresponding effect of (+)-(11 S ,12 S )-DB[ a,l ]P-dihydrodiol, the metabolic precursor of (+)- syn -(11 S ,12 R )-dihydrodiol (13 S ,14 R )-epoxide. After topical ap…

Stable Expression of Heterologous Sulfotransferase in V79 Cells: Activation of Primary and Secondary Benzylic Alcohols

Abstract A sulfotransferase (ST) capable of activating 1-hydroxymethylpyrene (HMP) and 9-hydroxymethylanthracene (HMA) to mutagens was purified from rat liver. This enzyme appeared to be identical with hydroxysteroid STa, whose cDNA was cloned and stably expressed in Chinese hamster V79 cells. Several primary and secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons induced gene mutations, sister chromatid exchanges (SCE) and/or cytotoxicity in these cells.

Fjord-region diol-epoxides of benzo[c]chrysene are potent inducers of micronuclei in murine bone marrow

Abstract Vicinal diol-epoxides are the best established carcinogenic metabolites of polycyclic aromatic hydrocarbons. Numerous studies have demonstrated their high genotoxic activity in various in vitro test systems. However, in vivo mutagenicity data are not available. The fjor-region diol-epoxides of benzo[ c ]chrysene combine high mutagenic activity in vitro with hydrolytic stability. They were tested for the induction of micronuclei in the bone marrow following intraperitoneal administration to NMRI mice. The anti diasteromer of the diol-epixode enhanced the frequency of micronucleated polycrhomatic erythrocytes strongly (7–19-fold above the value in untreated controls) over a very wide…

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…

Environmental Carcinogens: Polycyclic Aromatic Hydrocarbons. Von G. Grimmer. CRC Press, Boca Raton, FL, USA 1983. 261 S., geb. $ 81.50

Studies on Adduct Formation of (+)-Anti-Benzo[a]pyrene 7,8-Dihydrodiol 9,10-Epoxide with the oligonucleotides 5′-d(CCTATCGTTATCC) and 5′-d(CCTATm5CGTTATCC)

Abstract Adduct formation of (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide [BPDE] and 5′-d(CCTATCGTTATCC) or 5′-d(CCTATm5CGTTATCC) (G = binding target) has been studied. The extent of trans-BPDE-N2-dG adduct formation was higher in the oligonucleotide with 5′-d(m5CG) sequence context in both single- and double stranded form compared to the non-methylated analogue. The stimulating effect of m5dC on adduct formation has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring with the nucleobases prior to the nucleophilic addition. In the present study, both UV absorption and induced cir…

Structure elucidation of the adducts formed by fjord region Dibenzo[a,l]pyrene-11,12-dihydrodiol 13,14-epoxides with deoxyguanosine.

Model adducts to be used in the identification of biologically formed adducts were synthesized by reaction of fjord-region dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE) and deoxyadenosine (dA). The (+/-)-anti-DB[a,l]PDE was reacted with dA in dimethylformamide at 100 degrees C for 30 min to give four DB[a, l]PDE-14-N(6)dA adducts: (-)-anti-trans (26%), (+)-anti-trans (26%), (-)-anti-cis (17%), and (+)-anti-cis (17%). The (+/-)-syn-DB[a,l]PDE was reacted with dA under the same conditions to yield four DB[a, l]PDE-14-N(6)dA adducts and one N7Ade adduct: (+)-syn-cis (19%), (+)-syn-trans (13%), (-)-syn-cis (19%), (-)-syn-trans (13%), and (+/-)-syn-DB[a,l]PDE-14-N7Ade (22%). T…

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…

Glutathione Transferase A1-1 Catalyzed Conjugation of Polycyclic Aromatic Hydrocarbon Diol-Epoxides with Glutathione

Abstract The glutathione transferase A1-1 (GSTA1-1) isoenzyme catalyzes the formation of GSH-conjugates of the isomeric bay-region diol-epoxides (DEs) of trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (CDE) and trans-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene (DBADE) as well as the isomeric fjord-region DEs trans-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BPhDE) and trans-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydro-benzo[c]chrysene (BCDE) although with an approx. 20-fold variation in catalytic efficiency. With the anti-diastereomers and the syn-diastereomers of BPhDE and BCDE, GSTA1-1 demonstrated a significant preference for the enan…

Detoxication of carcinogenic fjord-region diol epoxides of polycyclic aromatic hydrocarbons by glutathione transferase P1-1 variants and glutathione.

AbstractEpidemiological studies suggest that individuals differing in the expression of allelic variants of the human glutathione transferase (GST) Pi gene differ in susceptibility to chemical carcinogens such as polycyclic aromatic hydrocarbons (PAH). This study reports the catalytic efficiencies (kcat/Km) of two naturally occurring variants, GSTP1-1/I-105 and GSTP1-1/V-105, towards a series of fjord-region diol epoxides representing potent biologically active PAH metabolites, and two GSTP1-1 mutants with Ala105 and Trp105 in the active site. The results indicate that individuals who are homozygous for the allele encoding GSTP1-1/V-105 might be more susceptible to PAH carcinogenesis due to…

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…

2,9-Dimethylpicene: Synthesis, Mutagenic Activity, and Identification in Natural Samples

Abstract 2,9-Dimethylpicene (2,9-DMPic) has been conveniently synthesized via a new route involving oxidative photocyclization of suitable substituted diarylethylenes and has been characterized by high-resolution 400-MHz 1H-NMR spectroscopy. Furthermore, 2,9-DMPic exhibits a typical line narrowed emission spectrum in n-decane matrix frozen at 15 K (Shpol'skii effect). Its unambiguous identification in a natural sample has been performed by this technique, providing strong evidence for the formation of this compound through aromatization of triterpenoid natural precursors. The mutagenicity of this naturally occurring methylated polycyclic aromatic hydrocarbon (PAH) has been examined in six h…

Species-dependent Metabolism of Benzo[c]phenanthrene and Dibenzo[a, l]pyrene by Various CYP450 Isoforms

Abstract The metabolism of benzo[c]phenanthrene (B[c]Ph) and dibenzo[a, l]-pyrene (DB[a, l]P) with various CYP isoforms including rat 1A1, 1A2, 2B1, 2E1, human 1A1, 1A2, 1B1, 2A6, 3A4, 2E1 and fish 1A expressed in Chinese hamster V79 cells has been compared. Major differences in the catalytic activities and in the regioselectivity of the eleven CYP isoforms with B[c]Ph and DB[a, l]P as substrates have been observed. There have been found substantially species-specific differences between homologous CYP isoforms at least when human, rat and fish are compared, which have to be taken into account when animal experiments are extrapolated to human. In particular, complementary catalytic activiti…

Studies of the binding of diolepoxide metabolites of polycyclic aromatic hydrocarbons to DNA using electrofluorescence polarization spectroscopy

In the electrofluorescence method, a solution of DNA with covalently bound polycyclic hydrocarbons is placed in an electric field, and changes in the intensity of polarized fluorescence are observed. Under the correct conditions, these charges can be used to determine a value for the angle psi between the long axis of the hydrocarbon molecule and the axis of the DNA helix. For DNA or poly(dA-dT) treated with each stereoisomer of anti-benzo[c]phenanthrene diolepoxide, psi ranged from 55 degrees to 61 degrees, consistent with a mixture of quasi-intercalated adenine adducts and externally bound guanine adducts. Similar results were obtained with another set of 'fjord-region' diolepoxides, deri…

Influence of aryl hydrocarbon- (Ah) receptor and genotoxins on DNA repair gene expression and cell survival of mouse hepatoma cells

The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called "bulky" DNA adducts including those generated by enzymatically activated B[a]P. Therefore, t…

Differential Enantioselectivity of Murine GlutathioneS-Transferase Isoenzymes in the Glutathione Conjugation ofTrans-3,4-dihydroxy-1,2-oxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene Stereoisomers

Abstract The kinetics of the glutathione (GSH) conjugation of (+)- and (−)-enantiomers ofanti- as well assyn-3,4-dihydroxy-1,2-oxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PDE) catalyzed by murine GSHS-transferase (GST) isoenzymes has been investigated. Murine GSTs exhibited significant differences in their enantioselectivity toward B[c]PDE stereoisomers. For example, while pi class isoenzyme mGSTP1-1 was virtually inactive toward stereoisomers with 1Sconfiguration [(−)-syn-and (+)-anti-B[c]PDE], these stereoisomers were good substrates for alpha class isoenzyme mGSTA1-2. When GST activity was measured as a function of varying B[c]PDE concentration (10–320 μM) at a fixed saturating conce…

Rat liver endothelial and Kupffer cell-mediated mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1.

The ability of isolated rat liver endothelial and Kupffer cells to activate benzo(a)pyrene (BP), trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (DDBP), trans-1,2-dihydroxy-1,2-dihydrochrysene (DDCH), and aflatoxin B1 (AFB1) to mutagenic metabolites was assessed by means of a cell-mediated bacterial mutagenicity assay and compared with the ability of parenchymal cells to activate these compounds. Endothelial and Kupffer cells from untreated rats were able to activate AFB1 and DDBP; DDBP was activated even in the absence of an NADPH-generating system. Pretreating the animals with Aroclor 1254 strongly enhanced the mutagenicity of the dihydrodiol, whereas the mutagenicity of AFB1 showed a sligh…

ChemInform Abstract: Synthesis of Oligodeoxynucleotides Containing Diastereomeric Dihydrodiol Epoxide-N6-Deoxyadenosine Adducts of Polycyclic Aromatic Hydrocarbons.

Abstract A generally applicable route is reported for the synthesis of oligodeoxynucleotides which contain structurally defined N 6 -deoxyadenosine adducts, derived from sterically highly hindered dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAH).

Synthesis of fjord region tetraols and their use in hepatic biotransformation studies of dihydrodiols of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene

Metabolic activation of the racemic benzo[c]chrysene-trans-9,10-, benzo[g]chrysene-trans-11,12- and dibenzo[a,l]pyrene-trans-11,12-dihydrodiols to fjord region syn- and anti-dihydrodiol epoxides by microsomes of Aroclor 1254-treated Sprague-Dawley rats has been examined. Since the fjord region dihydrodiol epoxides were hydrolytically unstable under the experimental conditions, their enzymatic formation was determined by analyzing the tetraols as their products of acidic hydrolysis upon addition of perchloric acid. The various stereoisomeric tetraols formed were separated by HPLC and identified by co-chromatography with authentic tetraols, which had been prepared by acidic hydrolysis of synt…

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…

A comparative investigation of DNA adducts, DNA strand breaks and gene mutations induced by benzo[a]pyrene and (+/-)-anti-benzo[a]pyrene-7,8-diol 9,10-oxide in cultured human cells.

Abstract Genotoxic effects of benzo[ a ]pyrene (BP) and its reactive metabolites (±)- anti -benzo[ a ]pyrene-7,8-diol 9,10-oxide ((±)- anti -BPDE) were comparatively investigated in vitro with the permanent human fibroblast cell line MRC5CV1. Induced DNA adducts were measured by 32 P-postlabeling, DNA strand breakage was determined by the comet assay and the HPRT gene mutation test was used to detect cytotoxicity and mutagenicity. Treatment of MRC5CV1 cells with S9 mix-activated BP or with (±)- anti -BPDE resulted in a concentration-dependent increase in DNA adducts and strand breaks. Genotoxic effects of BP and (±)- anti- BPDE were detected by 32 P-postlabeling and the comet assay with sim…

Relationship of Dibenzo[a, l]pyrene-DNA Binding to the Induction of p53, p21WAFIand Cell Cycle Arrest in Human Cells in Culture

Abstract The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the p21 WAFI protein which is involved in the mediation of growth arrest after DNA damage has occured. The exceptionally potent carcino-genic polycyclic aromatic hydrocarbon (PAH) dibenzo[a, l]pyrene (DB[a, l]P) and its ultimate metabolites, the fjord region (+)-syn- and (-)-anti-11,12-diol 13,14-epoxides (DB[a, l]PDE), were used in order to investigate DNA damage via adduct formation, subsequent induction of p53 and p21 WAFI , and cell growth behavior in human mammary carcinoma MCF-7 cells. Exposure of MCF-7 cells to 0…

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,…

Metabolism of 3-hydroxychrysene by rat liver microsomal preparations

3-Hydroxychrysene, a metabolite of the polycyclic aromatic hydrocarbon (PAH) chrysene, was metabolised by rat liver microsomal preparations obtained from Arochlor 1254-pretreated rats. Eight major metabolites were isolated by high performance liquid chromatography and characterised by u.v. spectroscopy and a variety of mass spectrometric techniques. The metabolites were unambiguously identified as 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene and 9-hydroxy-r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene and tentatively identified as 3-hydroxy-trans-5,6-dihydroxy-5,6-dihydrochrysene (since chrysene is a symmetrical molecule the 3- and 9-positions are equivalent), 9-hydroxy-trans-…

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 …

Detection of DNA effects in human cells with the comet assay and their relevance for mutagenesis

The single cell gel test (SCG-test or comet assay) is a rapid and sensitive method for measuring DNA damage and repair in individual cells. A wide variety of mutagens have been shown to cause DNA alterations detectable with the comet assay, but it is not yet clear whether a relationship exists between the DNA effects and the induction of mutations. We are therefore investigating in a cell culture system with human cells (MRC5CV1) the induction of DNA damage by environmental mutagens and the formation of mutations at the HPRT gene. In the present study we investigated benzo[a]pyrene (BP), an environmental mutagenic and carcinogenic polycyclic aromatic hydrocarbon, and its reactive metabolite…

Metabolic Activation of the (+)-S,S- and (−)-R,R-Enantiomers of trans-11,12-Dihydroxy-11,12-dihydrodibenzo[a,l]pyrene:  Stereoselectivity, DNA Adduct Formation, and Mutagenicity in Chinese Hamster V79 Cells

Polycyclic aromatic hydrocarbons require metabolic activation in order to exert their biological activity initiated by DNA binding. The metabolic pathway leading to bay or fjord region dihydrodiol epoxides as ultimate mutagenic and/or carcinogenic metabolites is thought to play a dominant role. For dibenzo[a,l]pyrene, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon, the formation of the fjord region syn- and/or anti-11,12-dihydrodiol 13,-14-epoxide (DB[a,l]PDE) diastereomers has been found to be the principal metabolic activation pathway in cell cultures leading to DNA adducts. In order to further elucidate the stereoselectivity involved in this activation pathway…

Book Review: Environmental Carcinogens: Polycyclic Aromatic Hydrocarbons. By G. Grimmer

Synthesis of oligodeoxynucleotides containing diastereomeric dihydrodiol epoxide-N6-deoxyadenosine adducts of polycyclic aromatic hydrocarbons

Abstract A generally applicable route is reported for the synthesis of oligodeoxynucleotides which contain structurally defined N 6 -deoxyadenosine adducts, derived from sterically highly hindered dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAH).

Malignant transformation of the liver tumour precursor cell line OC/CDE 22 by the four stereoisomeric fjord region 3,4-dihydrodiol 1,2-epoxides of benzo[c]phenanthrene

In previous work we established the rat liver oval cell line OC/CDE 22 in order to study in vitro mechanisms of liver cell transformation. We have now exposed OC/CDE 22 cells to each of the four optically active fjord region dihydrodiol epoxides of benzo[c]phenanthrene to investigate their capacity for malignant transformation of liver cells. All four configurational isomers, which are among the most potent carcinogenic metabolites of polycyclic aromatic hydrocarbons tested in murine tumour models, malignantly transform OC/CDE 22 cells at a 2 microM dose level, resulting in a similar colony-forming efficiency in soft agar. Inoculation of the transformed cells into newborn syngeneic rats pro…

Glutathione Conjugation of Bay- and Fjord-Region Diol Epoxides of Polycyclic Aromatic Hydrocarbons by Glutathione Transferases M1-1 and P1-1

Metabolism of polycyclic aromatic hydrocarbons in mammalian cells results in the formation of vicinal diol epoxides considered as ultimate carcinogens if the oxirane ring is located in a bay- or fjord-region of the parent compound. In the present study, individual stereoisomers of the bay-region diol epoxides of chrysene, dibenz[a,h]anthracene, and benzo[a]pyrene as well as of the fjord-region diol epoxides of benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]-chrysene have been incubated with GSH in the presence of human glutathione transferases GSTM1-1 (a mu-class enzyme) and GSTP1-1 (a pi-class enzyme). As previously shown with GSTA1-1 (an alpha-class enzyme) both M1-1 and P1-1 demonst…

Complex Metabolic Activation Pathways of Polycyclic Aromatic Hydrocarbons: 3-Hydroxy-trans-7,8-Dihydroxy-7,8-Dihydrobenzo[a]Pyrene as a Proximate Mutagen of 3-Hydroxybenzo[a]Pyrene

3-Hydroxybenzo[a]pyrene (3-OH-BP) is a major metabolite of benzo[a]pyrene (BP) in various systems. Metabolites of 3-OH-BP, formed by liver enzymes, bind to DNA1,2 and are mutagenic3,4. However, the active species have not yet been identified. Administration of 3-OH-BP to rats results in the excretion of sulfate and glucuronic acid conjugates of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol) (Fig. 1) as major metabolites in the bile5. The hydroxyl groups of this triol are structurally superimposable to those of 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene (9-hydroxychrysene-1,2-diol, Fig. 1), which is a metabolite of chrysene6,7 and a potent promutagen8,9. 9-…

CARCINOGENESIS: Glutathione S-transferase A1–1-catalysed conjugation of bay and fjord region diol epoxides of polycyclic aromatic hydrocarbons with glutathione

the fjord region diol epoxides a similar substrate enantioselectivity was noted, i.e. the enantiomer with the corresponding R configuration was again preferentially conjugated. In contrast, for the bay region syn -diol epoxides this substrate selectivity was reversed, resulting in a preference for the enantiomer with the S configuration. The chemically more reactive syn diastereomers were in general better substrates for GST Al-1 than the corresponding anti diastereomers. However, a comparison between different diol epoxide diastereomers revealed no obvious correlation between chemical reactivity of the compounds and catalytic efficiencies. Furthermore, no significant correlation between di…

Correlation of the Extent of Fjord-Region Oxidation with DNA Binding and Mutagenicity of the Enantiomeric 11,12-Dihydrodiols of Dibenzo[a,l]pyrene

Abstract In vitro studies on the hepatic biotransformation of the enantiomeric trans-11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) using microsomal fractions of animals pretreated with Aroclor 1254 revealed that the formation of fjord-region dihydrodiol epoxides strongly depends on the absolute configuration of the substrate. Both the (-)-11R,12R- and the (+)-11S,12S-enantiomer are converted diastereoselectively to the (-)- and (+)-anti-dihydrodiol epoxide, respectively, by either rat or mouse liver microsomes. Fjord-region oxidation occurs to greatest extent on incubation of the (-)-11R,12R-dihydrodiol with preparations from rats. This finding is in line with the differences seen for…

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…

Sulfotransferase-mediated chlorination of 1-hydroxymethylpyrene to a mutagen capable of penetrating indicator cells.

Methylated polycyclic aromatic hydrocarbons are common in the human environment. Many of them are stronger carcinogens than their purely aromatic congeners. They may be metabolized to benzylic alcohols. We report here on biochemical and toxicological characteristics of 1-hydroxymethylpyrene (HMP), a typical representative of this class of compounds. Rat liver cytosol, fortified with 3'-phosphoadenosine-5'-phosphosulfate, converted HMP into its sulfate ester (HMPS), HMPS bound covalently to isolated DNA. In physiological buffer at 37 degrees C, HMPS had a half-life of 2 min, the major decomposition product being HMP. Thus, cyclic activation is possible. When Cl- anions were present at physio…

Bestandsaufnahme zur Frage des Zusammenhangs zwischen dem DNA-Adduktgehalt in weißen Blutzellen und in humanem Lungengewebe

Genotoxicity characteristics of reverse diol-epoxides of chrysene.

Trans-3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol), a major metabolite of chrysene, is further metabolized by rat liver enzymes to products which effectively revert the his- Salmonella typhimurium strain TA98 to histidine prototrophy, but are only weakly mutagenic in strain TA100 and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). The liver enzyme mediated mutagenicity of chrysene-3,4-diol is substantially enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase. The predominant metabolites of chrysene-3,4-diol, namely the anti- and syn-isomers of its 1,2-oxide (termed reverse diol-epoxides), proved to be …

Glutathione conjugation of trans-3,4-dihydroxy 1,2-epoxy l,2,3,4-tetrahydrobenzo[c]phenanthrene isomers by human glutathione transferases

Each of the four stereoisomers of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene [(+)- and (-)-anti-BPhDE and (+)- and (-)-syn-BPhDE] has been incubated with the human glutathione transferase (GST) isoenzymes GST A1-1, GST M1-1 and GST P1-1, representing class alpha, mu and pi respectively, and glutathione (GSH). The conjugates formed were analyzed by HPLC and the results demonstrate that all GST isoenzymes catalyze the formation of GSH conjugates of all BPhDE isomers. However, a marked variation in catalytic efficiencies was observed (0.122-1.28/mM/s). These values are considerably lower than those previously estimated for the bay-region diol epoxides of benzo[a]pyren…