Efficient resolution of the dihydrodiol derivatives of benzo[a]pyrene by high-pressure liquid chromatography of the related (−)-dimenthoxyacetates

The diastereomeric (−)-dimenthoxyacetate derivatives of (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene were efficiently resolved by high-pressure liquid chromatography (HPLC) on silica gel. Treatment with methanolic ammonia under mild conditions removed the menthoxyacetate groups to furnish the optically pure (+) and (−) enantiomers of the trans-dihydrodiol. Epoxidation of each of the latter with m-chloroperbenzoic acid gave the corresponding (−) and (+) anti-diolepoxides. The speed and efficiency of resolution of these relatively sensitive compounds by HPLC demonstrates the potential utility of this technique for resolution of all types of carcinogen-derived arene dihydrodiols.     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

New procedure of high-voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids.

Addition of primary organic amines, such as n-butylamine, to the mobile phase altered the capacity factors and selectivity of benzo[a]pyrene metabolites obtained with reverse-phase high pressure liquid chromatography on an ODS column. Separation of benzo[a]pyrene phenols in particular was improved with 8 of the 10 available metabolites resolved, including those known to be biologically produced. The method offers sufficiently improved resolution or convenience that it should prove useful in comparative studies of metabolism of benzo[a]-pyrene and other polynuclear aromatic hydrocarbons. Applying the method to analysis of benzo[a]pyrene metabolites produced in vitro by hepatic microsomes from the marine fish Stenotomus versicolor indicated the principal phenolic derivatives produced by this fish were 1-hydroxy-, 3-hydroxy-, 7-hydroxy-, and 9-hydroxybenzo[a]pyrene.     

A benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide is the major metabolite involved in the binding of benzo[a]pyrene to DNA in isolated viable rat hepatocytes

Benzo[a]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[a]pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[a]pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[a]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.     

Excision repair by human fibroblasts of DNA damaged by r-7, t-8-dihydroxy-t-9,10-OXY-7,8,9,10-tetrahydrobenzo(a)pyrene

Benzo(a)pyrene diol-epoxide I (r-7,t-8,dihydroxy-t-9,10 oxy-7,8,9,10 tetrahydrobenzo(a)pyrene) was used to treat either human adenovirus 5 or cultures of human fibroblasts. The survival of diol-epoxide I treated adenovirus was greater when infecting fibroblasts from normal persons than when infecting fibroblasts from patients with xeroderma pigmentosum (XP). One diol-epoxide I molecule bound per viral genome correlated with one lethal hit as measured using XP fibroblasts.Normal fibroblasts blocked in semi-conservative DNA synthesis incorporated into their DNA more [³H]thymidine in response to diol-epoxide I treatment than did XP fibroblasts, and also excised more diol-epoxide I from their DNA. All of the effects described above were similar to those obtained when the inactivating agent was ultraviolet light rather than benzo(a)pyrene diol-epoxide I.     

Synthesis of the manganese-monocarbollide dianion [1-NHBu-2,2,2-(CO)3-closo-2,1-MnCB10H10]: Reactions with transition metal cations

Reaction of [Mn(NCMe)₃(CO)₃][PF₆] with Li₃[7-NHBu^t-nido-7-CB₁₀H₁₀] in THF (THF = tetrahydrofuran) affords the twelve-vertex manganacarborane dianion [1-NHBu^t-2,2,2-(CO)₃-closo-2,1-MnCB₁₀H₁₀]²⁻, isolated as the bis-[N(PPh₃)₂]⁺ salt (5a). This species reacts with {Pt(dppe)}²⁺ (dppe = Ph₂PCH₂CH₂PPh₂) to afford the bimetallic complex [1-NH₂Bu^t-2,3-{Pt(dppe)}-2,2,2-(CO)₃-closo-2,1-MnCB₁₀H₉] (7) which has an Mn-Pt bond. In contrast, with {Cu(PPh₃)}⁺ the anion of 5a yields a CuMnCu trimetallic compound [1-{NH(Bu^t)Cu(PPh₃)}-2,3,7-{Cu(PPh₃)}-3,7-(μ-H)₂-2,2,2-(CO)₃-closo-2,1-MnCB₁₀H₈] (8) in which one of the Cu centers is bonded to Mn, whilst the other is attached to the pendant NHBu^t group. Upon treatment with Ag⁺, compound 5a is oxidized giving the very unusual Mn(III)-carbonyl complex [1,2-μ-NHBu^t-2,2,2-(CO)₃-closo-2,1-MnCB₁₀H₁₀] (9a) in which the carborane ligand formally acts as an eight-electron donor to manganese. The novel structural features of compounds 7, 8, and 9a have been confirmed by X-ray diffraction studies.     

The kinetics of benzo(a)pyrene anti-7,8-dihydrodiol 9, 10-epoxide formation from benzo(a)pyrene and regulatory membrane effects

r-7,c-10,t-8,t-9-Tetrahydroxybenzo(a)pyrene (7,10/8,9-tetrol), which is the principal hydrolysis product of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-diol-epoxide), was resolved and measured by HPLC in organic extracts of incubations which contained induced rat liver microsomes and BP. Kinetic analyses showed that: (a) following a 5- to 7-min lag period, anti-diol-epoxide formation was linear, and (b) levels of anti-diol-epoxide formed were highly dependent upon the starting BP concentration. anti-Diol-epoxide production increased at starting BP concentrations of 0–12 μm and decreased in incubations containing 12–25 μm BP. However, between 25 and 100 μm BP, anti-diol-epoxide formation was stable at a level representing 65% of the peak production which occurred at a starting BP concentration of 12 μm. BP oxidation was competitively inhibited by (?)-trans-BP-7,8-dihydrodiol and about five times less effectively by the (+)-trans-BP-7,8-dihydrodiol. The inability of a severalfold excess of BP (25–100 μm) to totally inhibit BP-7,8-dihydrodiol oxidation was explained by the presence of a microsomal substrate compartment which was saturated at only 6–8 μm BP, the remaining BP present as aggregates in the aqueous compartment. Purification of microsomes by Sepharose 2B gel filtration after reaction with [³H]BP also indicated that BP-7,8-dihydrodiol was preferentially concentrated in the microsome compartment leading to a net increase in the ratio of BP-7,8-dihydrodiol to BP in the microsomal compartment, which favored BP-7,8-dihydrodiol oxidation to yield the biologically active anti-diol-epoxide.     

Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Indolylmethylene benzo[h]thiazolo[2,3-b]quinazolinones: Synthesis,characterization and evaluation of anticancer and antimicrobial activities

A series of novel 10-((1H-indol-3-yl)methylene)-7-aryl-7,10-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9(6H)-ones (8a–t) have been synthesized in good yields by the reaction of benzo[h]quinazoline-2(1H)-thiones (4a–f) with 2-chloro-N-phenylacetamide (5) followed by Knoevenagel condensation with various indole-3-carbaldehydes (7a–d) under conventional method. All the synthesized compounds were characterized by spectral studies and screened for their in vitro anticancer and antimicrobial activities. Compound 8c has exhibited excellent activity against MCF-7 (breast cancer cell line) than the standard drug Doxorubicin. Compound 8d against both the cancer cell lines, 8q against MCF-7 and 8c, 8h against HepG2 have also shown good activity. Remaining compounds have shown moderate activity against both the cell lines. Antimicrobial activity revealed that, the compound 8q and 8t against Staphylococcus aureus and 8i, 8k, 8l, 8q & 8t against Klebsiella pneumoniae have shown equipotent activity on comparing with the standard drug Streptomycin. Remaining compounds have shown significant antibacterial and comparable antifungal activities against all the tested microorganisms.     

An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish Stenotomus chrysops

Hepatic microsomal cytochrome P-450 from the untreated coastal marine fish scup, Stenotomus chrysops, was solubilized and resolved into five fractions by ion-exchange chromatography. The major fraction, cytochrome P-450E (M_r = 54,300), was further purified to a specific content of 11.7 nmol heme/mg protein and contained a chromophore absorbing at 447 nm in the CO-ligated, reduced difference spectrum. NH₂-terminal sequence analysis of cytochrome P-450E by Edman degradation revealed no homology with any known cytochrome P-450 isozyme in the first nine residues. S. chrysops liver NADPH-cytochrome P-450 reductase, purified 225-fold (M_r = 82,600), had a specific activity of 45–60 U/mg with cytochrome c, contained both FAD and FMN, and was isolated as the one-electron reduced semiquinone.Purified cytochrome P-450E metabolized several substrates including 7-ethoxycoumarin, acetanilide, and benzo[a]pyrene when reconstituted with lipid and hepatic NADPH-cytochrome P-450 reductase from either S. chrysops or rat. The purified, reconstituted monooxygenase system was sensitive to inhibition by 100 μM 7,8-benzoflavone, and analysis of products in reconstitutions with purified rat epoxide hydrolase indicated a preference for oxidation on the benzo-ring of benzo[a]pyrene consistent with the primary features of benzo[a]pyrene metabolism in microsomes. Cytochrome P-450E is identical to the major microsomal aromatic hydrocarbon-inducible cytochrome P-450 by the criteria of molecular weight, optical properties, and catalytic profile. It is suggested that substantial quantities of this aromatic hydrocarbon-inducible isozyme exist in the hepatic microsomes of some untreated S. chrysops. The characterization of this aryl hydrocarbon hydroxylase extends our understanding of the metabolism patterns observed in hepatic microsomes isolated from untreated fish.     

Determination of albumin adducts of (+)-anti-benzo[a]pyrene-diol-epoxide using an high-performance liquid chromatographic column switching technique for sample preparation and gas chromatography–mass spectrometry for the final detection

A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC–MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r²=0.99 and r²=0.98 for BT I-1 concentration ranges of 4–400 ng/l and 4–40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.     

Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[a]pyrene (BaP), benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11H-benzo[b]fluorene, dibenz[a,h]anthracene, pyrene, benzo[ghi]perylene and benzo[e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer.The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.     

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives

To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[a]pyrene (B[a]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[a]P, 6-acetoxymethyl(6-AcOCH₂)B[a]P, 6-hydroxymethyl(6-HOCH₂)B[a]P, 6-methyl(6-CH₃)B[a]P and 6-benzoyloxymethyl(6-BzOCH₂)B[a]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH₂)B[a]P were less active, and 6-methoxymethyl (6-MeOCH₂)B[a]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH₂- = 6-HOCH₂- = or > B[a]P > 6-CH₂- > 6-BzOCH₂- > 6-ClCH₂- > 6-OCH- > 6-BrCH₂B[a]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH₂-, 6-BzOCH₂- and 6-OCHB[a]P to 6-HOCH₂B[a]P. The halomethyl compounds were converted to 6-HOCH₂B[a]P in the absence of enzyme by hydrolysis. 6-MeOCH₂B[a]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[a]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH₂-derivatives are carcinogenic.     

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major β-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the β-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrodrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.     

Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.     

The metabolism of 7- and 12-methylbenz[a]-anthracene and their derivatives

1. 7- and 12-Methylbenz[a]anthracene were converted by rat-liver homogenates into the corresponding hydroxymethyl derivatives, products that are probably the 8,9-dihydro-8,9-dihydroxy and the 5,6-dihydro-5,6-dihydroxy derivatives, and a number of phenolic products. 2. Both hydrocarbons were converted into glutathione conjugates; that from 7-methylbenz[a]anthracene was also formed, together with 5,6-dihydro-5,6-dihydroxy- and 5-hydroxy-benz[a]anthracene, from 5,6-epoxy-5,6-dihydro-7-methylbenz[a]anthracene. 3. 7- and 12-Hydroxymethyl-benz[a]anthracene were converted into products that are probably 8,9-dihydro-8,9-dihydroxy derivatives, and into phenols. 4. The preparation of a number of derivatives of the hydrocarbons is described. 5. The oxidation of the hydrocarbons with lead tetra-acetate was investigated.     

Formation of DAN-binding products from isolated benzo[a]pyrene metabolites in rat liver nuclei

Liver nuclei from 3-methylcholanthrene-treated rats in the presence of NADPH metabolized 3- and 9-hydroxybenzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to products that bound to DNA. Maximal binding was obtained with the dihydrodiol which was approximately 3-fold that with 9-hydroxybenzo[a]pyrene, and 60-fold that with 3-hydroxybenzo[a]pyrene, as substrates. Both 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene and 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene were also extensively metabolized by the nuclear fraction but did not give rise to DNA-binding products.The available evidence suggests that the DNA binding species derived from 9-hydroxy-benzo[a]pyrene is 9-hydroxy-benzo[a]pyrene-4,5-oxide and from 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, as previously observed in different systems, 7,8-dihydro-7,8-dihydroxy-benzo[a]pyrene-9,10-oxide.     

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0 mg/kg. Lungs and livers were harvested after 24 h, the DNA extracted and subjected to ³²P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50 mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.