Epoxide Of Benzo Research Articles

Differences in detoxification of the ultimate carcinogenic dihydrodiol epoxides of benzo[a]pyrene and dibenzo[a,l]pyrene in human Caco-2 cells

Differences in detoxification of the ultimate carcinogenic dihydrodiol epoxides of benzo[a]pyrene and dibenzo[a,l]pyrene in human Caco-2 cells

Analysis of GSH Conjugates of Bay- and Fjord-Region Dihydrodiol Epoxides of Benzo[a]pyrene and Dibenzo[a,l]pyrene and their Transport in Enterocyte-like Caco-2 Cells

The gastrointestinal tract is a main route of exposure to polycyclic aromatic hydrocarbons (PAH) due to the ingestion of PAH-contaminated food. Numerous PAH are carcinogenic and most of them are metabolized to biologically active bay- and fjord-region dihydrodiol epoxides that are capable to form stable DNA adducts leading subsequently to mutations and cell transformation. In vitro studies with canine kidney epithelial cells demonstrate that detoxification of dihydrodiol epoxides through glutathione (GSH) conjugate formation catalyzed by glutathione S-transferases and their subsequent removal from the cell by transport proteins of the ATP-binding cassette superfamily is an important process to control DNA adduct levels. In the present study, Caco-2 cells were selected to serve as a model system to investigate both the human intestinal metabolism of the carcinogenic PAH benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and transport of their conjugates. In order to allow application of non labeled compounds specific LC-ESI-MS/MS methods were established to determine GSH conjugate formation of the dihydrodiol epoxides of BP and DBP and to demonstrate their efflux from Caco-2 cells.

The role of hydrogen bonding in some diol epoxides

The role of intramolecular hydrogen bonding in determining the properties of two diol epoxides of benzene has been investigated by means of ab-initio SCF calculations (Gaussian 70, STO-5G). These two systems were used as models for the syn and anti 7,8-diol-9,10-epoxides of benzo[a]pyrene, one or both of which are believed to be ultimate carcinogenic forms of benzo[a]pyrene. It was found that there is intramolecular hydrogen bonding in the syn diol epoxide of benzene, but not the anti isomer. This hydrogen bonding was shown to have the effect of facilitating the opening of the epoxide ring at either carbon, but particularly at C(1). These findings are fully consistent with the observed properties of the previously mentioned diol epoxides of benzo[a] pyrene and help to explain these properties.

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane.

Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo[a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmol x min(-1) x pmol(-1) CYP1A1 of the enantiomeric promutagens (-)- and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both Vmax and Km values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1.

FT-Raman, FT-IR and normal-mode analysis of carcinogenic polycyclic aromatic hydrocarbons. Part II?a theoretical study of the transition states of oxygenation of benzo(a)pyrene (BaP)

The first process in polycyclic aromatic hydrocarbons (PAH) metabolism is believed to be the oxygenation that leads to several structural isomeric epoxides. Most of these intermediates will be converted into non-toxic hydrophilic metabolites and be excreted. Because of this, metabolism has been considered to be primarily a detoxification mechanism. However, some may be activated to become ultimate carcinogens. Knowledge of the identities of these intermediates and their probabilities of formation will be of vital importance in mechanism elucidation and cancer prevention. Theoretically, epoxidation of benzo(a)pyrene (BaP) can take place at any one of the sites: 1,2-, 2,3-, 4,5-, 7,8-, 8,9-, 9,10- and 11,12-, leading to seven possible isomeric epoxides. The conformations of these isomers formed from direct oxygenation were simulated and their activation energies calculated. Among them, the 4,5-epoxide appears to be the most and the 8,9-complex the least energetically favorable. The 11,12-, 9,10- and 7,8-isomers are almost equally energetic. The rate of formation of the 7,8-isomer, which is believed to be the precursory carcinogen, relative to the 4,5-epoxide is estimated to be 9.1 × 10−4 : 1. On the other hand, the relative rates of 1,2- and 2,3-complexes are extremely low. When the protoporphyrin group is used for simulation, the BaP plane is seen parallel to the porphyrin plane in the 1,2- and 2,3-complexes, but angled away to various degrees in all other epoxides. The parallel orientation results in a significant lowering of the activation energy, making 1,2- and 2,3-complexes the most favorable intermediates. This is in agreement with chromatographic analyses of BaP metabolism in liver microsomes. In these experiments, 3-hydroxy-BaP is the major product. Copyright © 2001 John Wiley & Sons, Ltd.

Bay-Region Diol Epoxides of Benzo[a]Pyrene are Stealth Poisons of Topoisomerase I

Topoisomerase I transiently cleaves one strand of duplex DNA to relieve torsional strain during enzymatic processing events such as transcription and replication. Thus, topoisomerase I is an important target for anti-tumor drugs. We have examined the effects of cis- and trans-opened (+)-7R,8S,9S,10R)- and (−)-(7S,8R,9R,10S)-benzo[a]pyrene 7,8-diol 9,10-epoxide adducts at the exocyclic amino groups of the purine bases at or near a known cleavage site in a 22-mer DNA duplex. The dG adducts lie in the minor groove either upstream or downstream from the modified base, and the dA adducts are intercalated at either side of the modified base. Thus four distinct structural motifs were available for study. The dG adducts inhibit cleavage at the normal site with resultant remote cleavages being observed. In contrast, three of the four dA adducts examined appear to be initially invisible to the enzyme since they allow the normal cleavage to occur, but they prevent subsequent religation and thus act as topoisomerase “stealth poisons”.

DNA Adducts Formed by the Fjord-Region Dihydrodiol Epoxide of Benzo[S]Picene

Anti benzo[s]picene 9,10-dihydrodiol 11,12-epoxide was reacted with calf thymus DNA and with its constituent deoxyribonucleotides. The origin of DNA adducts was established by comparison of their HPLC retention times and UV spectra with those of the nucleotide-derived adduct-markers. The low binding of the dihydrodiol epoxide to DNA (2%) and to deoxyguanylic and deoxyadenylic acids precluded an NMR study, and assignment of cis or trans epoxide ring opening was made by analogy to other anti dihydrodiol epoxide adducts. The adduct ratio for dGuo/dAdo in DNA was 24/76 and the ratios of dGuo cis/dGuo trans and dAdo cis/dAdo trans were 40/60 and 6/94, respectively.

Effects of Diol Epoxide Adducts on Binding of Different Transcription Factors to DNA

The (-)-anti-diol epoxide of benzo[c]phenanthrene (BPhDE) and (+)-anti-diol epoxide of benzo[a]pyrene (BPDE) were used to site-specifically modify oligonucleotides containing the binding site for the transcription factors AP-1 and TFIID, respectively. The modified oligonucleotides were incubated with the appropriate transcription factor and protein-DNA interaction analysed by the electrophoretic mobility shift assay. The available results show that a trans-BPhDE-N6-dA adduct located in the AP-1 binding sequence totally inhibits protein-DNA interaction. The presence of a trans-BPDE-N2-dG adduct near the TFIID binding site and thus, an expected bending of the helix, has no obvious effect on protein binding or the further assemblance of the transcription initiation complex.

Characterization of DNA Adducts Formed by the Four Configurationally Isomeric 5,6-Dimethylchrysene 1,2-Dihydrodiol 3,4-Epoxides

The DNA adducts formed from the racemic syn and anti dihydrodiol epoxides of 5,6-dimethylchrysene were characterized through various spectroscopic methods. Substantial reaction with the amino groups of both deoxyadenosine and deoxyguanosine residues were detected with both the syn and anti derivatives. The chemical shifts and coupling constants for the cis and trans opened adducts from the syn dihydrodiol epoxide were distinctly different, whereas for the anti dihydrodiol epoxide these properties were fairly similar for cis and trans adducts. In the latter case, assignment of trans and cis configurations was less obvious, and the finding that trans adducts have always predominated over cis adducts for all dihydrodiol epoxides studied to date was helpful in making these assignments. The preferential formation of cis adducts in DNA by the syn dihydrodiol epoxide is more like the chemistry of the dihydrodiol epoxide of benzo[c]phenanthrene than of benzo[g]chrysene, although both of these, like 5,6-dimethylchrysene, are non-planar compounds.

The Stereoisomeric Fjord-Region Benzo[c]phenanthrene-3,4-Dihydrodiol 1,2-Oxides Malignantly Transform Rat Liver Epithelial Cells

Abstract The rat liver oval cell line OC/CDE 22 was exposed 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 malignantly transformed OC/CDE 22 cells, resulting in a similar colony-forming efficiency in soft agar. Inoculation of the transformed cells into newborn syngeneic rats produced an extremely high incidence of carcinomas with a short latency period. The induced carcinomas displayed cholangiocellular, adenoid and solid growing structures. Thus, the fjord-region dihydrodiol epoxides of benzo[c]phenanthrene are able to efficiently transform rat liver cells. Furthermore, experimental evidence is presented that proliferation plays a central role in liver cell transformation mediated by polycyclic aromatic hydrocarbons.

Electrostatic effects on the rates of DNA-catalyzed reactions

Abstract Diol-epoxide metabolites of many genotoxic polycyclic aromatic hydrocarbons are hydrolyzed to tetraols in a detoxification reaction. The hydrolysis reaction has both spontaneous and acid-catalyzed components; moreover, the reaction rate increases in the presence of DNA. The best studied of these diol epoxide metabolites are the trans 7,8 diol-9,10 epoxides of benzo[a]pyrene: anti -BPDE, the proximate carcinogen, in which the oxirane ring is anti with respect to the 7-hydroxyl group, and its syn diastereomer, syn -BPDE. Jerina and coworkers have studied the kinetics of the hydrolysis of syn - and anti -BPDE as a function of pH and DNA concentration and have measured the equilibrium constant for the formation of a noncovalent complex with DNA. They constructed a two-state model in which the diol epoxide is either free or statically bound: the free fraction is hydrolyzed with the same kinetics as it exhibits in solution without DNA; the bound diol epoxide reacts at faster rates. In this model, the dependence of the observed hydrolysis rates on both DNA concentration and pH is explained by using the rate constants, k 0 and k H , for reactions of the free diol epoxide, the rate constants, k cat 0 and k cat H , for the bound molecule, and the binding constant, K eq . The present work uses an acidic-domain interpretation of the two-state model to explain the catalytic effect of DNA on the acid-catalyzed hydrolysis of syn - and anti -BPDE. Postulating that the rate enhancement is a result of acidic domains at the DNA surface, we assumed the relationship k cat H = k H [H + ] b , where [H + ] b is the hydrogen ion concentration near the bound molecule. Using numerical solutions to the Poisson-Boltzmann equation, the pH dependence of acidic domains at the surface of the polyelectrolyte, DNA, was calculated. Energy-minimization calculations were used to estimate the conformations of diol epoxide-DNA intercalation complexes. Poisson-Boltzmann (PB) calculations on these structures yielded hydrogen-ion concentrations near the epoxide group consistent with the k cat H /k H ratio over a range of added-salt concentrations. The results strongly suggest that DNA catalysis of diol-epoxide hydrolysis is a polyelectrolyte effect. The mechanisms and rate constants observed for the acid-catalyzed hydrolysis in the absence of DNA are consistent with the increase in the rate constant induced by DNA. It may be concluded that the catalysis is primarily an effect of the acidic domains in the surface grooves of the nucleic acid.

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 produced an extremely high incidence of carcinomas with a short latency period. The induced carcinomas displayed cholangiocellular, adenoid and solid growing structures. Neither cell growth in soft agar nor induction of tumor formation in newborn rats were achieved if confluent OC/CDE 22 cell cultures were exposed to each of the four stereoisomers and left in the confluent state for 4 weeks. In contrast, if confluent cells were exposed to the four stereoisomers, immediately split and then subcultured as usual, full transformation was accomplished. Our results indicate that the fjord region dihydrodiol epoxides of benzo[c]phenanthrene are highly efficient transforming agents for rat liver cells and that proliferation plays a pivotal role in the liver cell transformation process induced by polycylic aromatic hydrocarbons.

Modulation of transcription in rat liver nuclei in vitro by a diol epoxide of benzo[a]pyrene.

Treatment of isolated rat liver nuclei with 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene, the ultimate carcinogenic metabolite of benzo[a]pyrene, resulted in inhibition of transcription as measured by radioactive precursor incorporation into RNA. The mechanism of inhibition as analyzed by use of different types of inhibitors suggested that the carcinogen acted on both the major components of transcription machinery, that is, the template chromatin and the enzyme RNA polymerases. This action correlates well with the observations made after administration of benzo[a]pyrene to rats.

Linoleate-dependent co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by rat cytosolic lipoxygenase.

1. Co-oxygenation of 14C-labelled benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol was studied in rat lung cytosol, using linoleic acid as a co-substrate. Covalently bound and soluble metabolites were quantified by radiometry and h.p.l.c., respectively. 2. The co-oxygenation resulted in the production of reactive metabolites capable of protein binding as well as a series of soluble derivatives. 3. Co-oxygenation of benzo(a)pyrene yielded primarily a significant amount of benzo(a)pyrene-6,12-dione while benzo(a)pyrene-7,8-dihydrodiol led to a significant amount of benzo(a)pyrene-trans-anti-tetrol. 4. Their production was abolished by addition of 25 microM of the lipoxygenase inhibitor and antioxidant NDGA. 5. It is postulated that the linoleic acid peroxyl radicals, formed by rat lung lipoxygenase, initiate the one-electron oxidation of benzo(a)pyrene to its quinones, and epoxidation of benzo(a)pyrene-7,8-diol to the ultimate carcinogenic benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide.

Identification of (+) and (−) anti benzo[a]pyrene dihydrodiol epoxide–nucleic acid adducts by the 32P-postlabeling assay

Purine deoxyribonucleoside 3'-phosphates were reacted with the (+)- and (-)-enantiomers of the anti dihydrodiol epoxide of benzo[a]pyrene. Products from cis and trans opening of the epoxide ring were separated by HPLC and they were identified by comparison of their CD spectra with those known for the corresponding nucleoside adducts. Thereafter, the eight known benzo[a]pyrene-purine deoxyribonucleoside-3'-phosphate adducts were postlabeled with [32P]ATP and T4 kinase and the positions of these individual bisphosphates were mapped by TLC. Though all eight adducts migrated to the same general region of the thin layer plates, the four possible adducts from each enantiomeric dihydrodiol epoxide were resolved.

Co-oxidation of xenobiotics: Lipid peroxyl derivatives as mediators of metabolism

Systems which carry out peroxyl-dependent oxidations can serve as activation systems for carcinogenic compounds. Some function via classical peroxidase reactions in which an enzyme-derived oxidant performs the electron abstraction from or oxygen donation to the oxidizable substrate. This mechanism applies to the peroxidative activation of aromatic amines and of the phenolic compound diethylstilbestrol. These classical peroxidase reactions may be initiated by hydrogen peroxide or by organic peroxides, including lipid hydroperoxides. A different mechanism is involved in the oxygenation of polycyclic aromatic hydrocarbons and of aflatoxin B 1. In these cases the oxidant is a peroxyl radical, and the reaction occurs by the direct, non-enzymatic interaction of the peroxyl radical and the oxidizable substrate. Most peroxyl radicals in biological systems are lipid-derived. The key reaction which distinguishes the peroxyl radical-dependent oxidations from the classical peroxidase reactions is the ability of the former to epoxidize activated carbon-carbon double bonds. The epoxidation of benzo[ a]pyrene derivatives has been studied extensively in subcellular and whole cell and tissue systems, and is discussed as a model for this class of reaction. Determining the generality of this activation path and its role in vivo present the major questions to be answered in regard to the importance of these reactions in chemical carcinogenesis.

7,12-Dimethylbenz[a]anthracene: refined structure, electron density distribution and endo-peroxide structure.

The crystal structure of 7,12-dimethylbenz[a]anthracene (DMBA) has been refined from new X-ray diffraction data collected at low temperature (180 K). This has allowed the location of the hydrogen atom positions not previously reported in earlier structure determinations and refinements; a more precise molecular geometry is therefore now presented. In addition, an analysis of the electron density in this carcinogenic molecule has been made by multipole refinement. These two types of studies give information on the amount of strain in the bay region and the distribution of electron density in the molecule. The molecule is highly distorted in the bay region as a result of steric overcrowding between hydrogen atoms (minimum H ... H 2.06 A) so that torsion angles of 18 degrees and 22 degrees occur in this area. The bonds in the bay region and to the two methyl groups appear to be electron-rich; however, while the K-region of DMBA has a high pi-bond density computed from interatomic distances, the multipole analysis does not indicate that it is highly electron-rich. The 7- and 12-positions (equivalent to the 9- and 10-positions of anthracene) are highly reactive and appear to show a deficiency of electron density. Molecular dioxygen can add across these positions to give a peroxy compound. The crystal structure of such an endo-peroxide of DMBA has also been studied at 180 K although not to the high precision obtained for the parent compound. Some distortions are apparent in this molecule; in particular small CH3-C-O angles (101-104 degrees) are observed, indicative of some strain in the molecule. A computer graphics analysis of the diol epoxides of DMBA, generated from X-ray coordinates of DMBA and reported values for a diol epoxide of benzo[a]pyrene, show that steric overcrowding may affect the conformation of certain isomers of the diol epoxides.

Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya' and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diolepoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung GST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (pI 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia.

Identification of metabolites of benzo[b]fluoranthene.

The metabolism by rat liver 9000 x g supernatant of the environmental carcinogen benzo[b]fluoranthene was investigated. The major metabolites were identified, by comparison to synthetic samples, as 5- and 6-hydroxybenzo[b]fluoranthene and 4- and 7-hydroxybenzo[b]fluoranthene. The principal dihydrodiol metabolite formed under these conditions was trans-11,12-dihydro-11,12-dihydroxybenzo[b]fluoranthene, which was identified by comparison to the synthetic compound. 1,2-Dihydro-1,2-dihydroxybenzo[b]fluoranthene was identified, by its mass spectrum and by comparison if its u.v. spectrum to that of a synthetic model compound, 11-ethylidene-11H-benzo[b]fluorene. No evidence was obtained for the formation of 7b,8-dihydro-7,8-dihydroxybenzo[b]fluoranthene or trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene. The latter would be the precursor to a bay region dihydrodiol epoxide of benzo[b]fluoranthene.