Synthesis,in Vitro Metabolism,Mutagenicity, and DNA-Adduction of Naphtho[1,2- e ]pyrene

Literature data, although limited, underscore the contribution of C 24 H 14 polycyclic aromatic hydrocarbons to the biological activity of the extracts of complex environmental samples. However, little attention has been given to naphthopyrene (NP) isomers due to lack of synthetic standards and, therefore, lack of studies aimed at unequivocal identification of C 24 H 14 isomers in environmental samples. We hypothesize that naphtho[1,2- e ]pyrene (N[1,2- e ]P), having a fjord region and a bay region might be a potent mutagen. To test our hypothesis, we synthesized N[1,2- e ]P by Suzuki reaction and examined its in vitro metabolism with the S 9 fraction from phenobarbital/ g -naphthoflavone-induced rat liver homogenates. We have tentatively identified its K-region diol as the major metabolite and a fjord region dihydrodiol as minor metabolite. Contrary to our hypothesis, N[1,2- e ]P was not mutagenic in S. typhimurium strain TA 100 and did not induce morphological cell transformation in mammalian cells. Its metabolite, 11,12-dihydroxy-11,12-dihydro-N[1,2- e ]P was a weak mutagen. The reaction of calf thymus DNA with N[1,2- e ]P-11,12-diol-13,14-epoxide showed that it reacts predominately with the exocyclic amino group of adenine.     

DETERMINATION OF DIBENZO[A,L]PYRENE AND OTHER FJORD-REGION PAH ISOMERS WITH MW 302 IN ENVIRONMENTAL SAMPLES

Polycyclic aromatic hydrocarbons (PAHs) occur in the environment as complex mixtures including compounds with mutagenic and carcinogenic activity. The PAH profile routinely determined in environmental samples at present encompasses isomers with molecular weight (MW) not greater than 300. However, PAHs with MW >300 have been demonstrated for several matrices to contribute up to 50% of the total activity when tested for carcinogenicity in experimental animals. Recent studies indicate that among the dibenzopyrenes with MW 302 dibenzo[a,l]pyrene, possessing a fjord region, is by far the most carcinogenic PAH hitherto identified. To further elucidate the environmental relevance of this compound we have applied the isotope dilution GC/MS technique as analytical procedure to determine this compound and the related fjord region PAH naphtho[1,2-a]- and naphtho[1,2-e]pyrene in various matrices. Identification was based on comparison of UV and mass spectra as well as retention times of authentic reference materials. Determination of these PAHs was achieved after clean-up by several chromatographic steps including fractionation on a modified TABA-silica gel column. Quantitative data for matrices such as two cigarette smoke condensates, motor vehicle exhaust condensate (Otto-type engines), and tar-cork are reported. Based on toxic equivalent factors the relative contribution of dibenzo[a,l]pyrene (5.4–42.3%) to the total carcinogenic activity of a PAH profile will be discussed comprising 14 selected isomers (benzo[b]naphtho[2,1-d]thiophene; cyclopenta[cd]pyrene; benz[a]anthracene; chrysene/triphenylene; sum of benzo[b]-, benzo[k]-, and benzo[j]fluoranthene; benzo[a]pyrene; indeno[1,2,3-cd]pyrene; dibenz[a,h]anthracene; benzo[ghi]perylene; anthanthrene; dibenzo[a,l]pyrene determined in these matrices.     

Syntheses and Identification of Benzo[C]Chrysene Metabolites

Like other PAHs, chrysenes are thought to exert their carcinogenicity via metabolic activation of proximally carcinogenic dihydrodiols to diol epoxides as ultimate carcinogens. Benzo[c] chrysene (B[c]C) is structurally intriguing among the PAH because it features both a bay region and a fjord region. Although B[c]C is carcinogenic and mutagenic, few data are available on its metabolic activation or the nature of its metabolites.

We have synthesized the B[c]C trans-1,2-, 7,8-, and 9,10-dihydrodiols from the appropriate methoxy-substituted bisnaphthyl olefins by photochemical cyclization. B[c]C was metabolized with S9 liver fraction from phenobarbital/β-naphthoflavone-treated rats. Dihydrodiols were formed on both terminal rings as well as in the K-region. 2-, 3-, and 10-HydroxyB[c]C were also identified as metabolites. In mutagenicity studies toward S. typhimurium TA100, 1,2-dihydrodiol was more mutagenic than B[c]C at doses above 1.25 μg/plate, whereas 9,10-dihydrodiol was toxic at doses above 1.25 μg/plate.     

Evaluation of Benzo[c]Chrysene Dihydrodiols in the Morphological Cell Transformation of Mouse Embryo Fibroblast C3H10T1/2CL8 Cells

The morphological cell transforming activities of three dihydrodiols of benzo[c]chrysene (B[c]C), trans?B[c]C-7,8-diol, trans?B[c]C-9,10-diol, and trans?B[c]C-1,2-diol were compared to those of B[c]C in order to study the possible routes of metabolic activation in transformable C3H10T1/2 mouse embryo fibroblasts. B[c]C-treated C3H10T1/2 cells exhibited a concentration-related increase in morphologically transformed foci over a concentration range of 0–3 μg/ml. At 3 μg/ml, B[c]C induced 1.23 Type II & III foci/dish, with 73% of the dishes exhibiting Type II or Type III foci, and a survival of 87%. trans?B[c]C-7,8-diol produced concentration-related responses over a range of 0–5 μg/ml. At 3 μg/ml, trans?B[c]C-7,8-diol produced 1.13 Type II & III foci/dish with 72% of the dishes exhibiting foci, and a survival of 76%. trans?B[c]C-9,10-diol was inactive as a morphological cell transforming agent over a concentration range of 0–3 μg/ml. trans?B[c]C-1,2-diol was also inactive as a morphological cell transforming agent over a concentration range of 0–3 μg/ml. These results suggest that a K-region dihydrodiol of B[c]C, trans?B[cC-7,8-diol, may play a role in the ability of B[c]C to morphologically transform C3H10T1/2 cells.     

Identification of Stereochemical Configurations of Cyclopenta[ cd ]pyrene DNA Adducts in Strain A/J Mouse Lung and C3H10T1/2CL8 Cells

Four major and several minor DNA adducts were resolved by 32 P-postlabeling analysis of DNA from strain A/J mouse lung and C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts treated with cyclopenta[ cd ]pyrene (CPP). The identical pattern of adducts was seen in vivo and in vitro. Cochromatography of synthetic resolved diastereomers of cis - and trans - N 2 -CPP-deoxyguanosine-3'-phosphates with the in vivo adducts obtained from strain A/J mouse lung and in vitro adducts obtained from C3H10T1/2 cells allowed identification of the predominant DNA adduct as cis -(3 R ,4 S )- N 2 -CPP-deoxyguanosine. The second most abundant adduct formed in vivo and in vitro was identified as trans -(3 S ,4 S )- N 2 -CPP-deoxyguanosine.     

Biotransformation and DNA Adduct Formation of Trans-8,9-Dihydroxy-8,9-Dihydrodibenzo[a,l]Pyrene by Induced Rat Liver and Human CYP1A1 Microsomes

In order to explain the adduct patterns observed from the human CYP1A1-mediated binding of dibenzo[a, l]pyrene (DB[a, l]P) to DNA, we have investigated the further metabolism and DNA adduct activity of trans-DB[a, l]P-8,9-diol by induced rat liver and human CYP1A1 microsomes. trans-DB[a, l]P-8,9-diol was synthesized and metabolic studies with β-naphthoflavone-induced rat liver microsomes indicated three major metabolites: 2 diastereomers of trans,trans-8,9,11,12-tetrahydro-8,9,11,12-tetrahydroxy-DB[a, l]P and 8,9,13,14-tetrahydro-8,9,13,14-tetrahydroxy-DB[a, l]P. DB[a, l]P when activated by CYP1A1/epoxide hydrase (EH) and calf thymus DNA gave a complex pattern of DNA adducts most of which cochromatograph with syn- and anti-DB[a, l]P fjord region diol epoxide-DNA standards. Two highly polar eluting adducts were also observed, one which cochromatographs with the single major DNA adduct obtained from the CYP1A1/EH activation of trans-DB[a, l]P-8,9-diol. The relative retention time of this adduct suggests either a bis-diol epoxide adduct or a more polar diol epoxide adduct.     

Relationship Between Inhaled PAH and Urinary Excretion of Phenanthrene,Pyrene and Benzo[a]pyrene Metabolites in Coke Plant Workers

Abstract

By means of personal air samplers the exposure to polycyclic aromatic hydrocarbons (PAH) of four coke employees working at different locations was measured during 4 running days. Simultaneously the 24 hrs urines were collected. A simple, well reproducible method for the determination of 9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, 1-; 2-; 3-; 4- and 9-hydroxyphenanthrenes, 1,2-; 3,4-; and 9,10-dihydroxydihydrophenanthrenes as well as 1-hydroxypyrene and 1,2-dihydroxy-1,2-dihydropyrene was used. As expected, workers on the battery topside are most exposed and, accordingly, excrete by far the highest amount of PAH metabolites. A good correlation between the PAH inhaled during 8 hrs and the metabolites excreted in the 24 hrs urine is observed. 20% to 30% of the inhaled phenanthrene is excreted as dihydrodiols, but only 0.5% of the inhaled benzo[a]pyrene forms 9,10-dihydrodiol in the urine. The individual invariance of the metabolite profile of the isomeric phenols and dihydrodiols indicates a genetically caused enzyme pattern of oxygenases, which can or cannot be induced by exogenous factors. More investigations are necessary to clarify whether this metabolic profile may be suitable as a marker for carcinogenic risk.     

Species-Dependent Metabolism of Benzo[c]Chrysene Mediated by c-DNA-Expressed Human,Rodent and Fish Cytochrome P450 Enzymes

The metabolism of benzo[c]chrysene (B[c]Ch) with various cytochrome P450 (CYP) enzymes including rat 1A1, 1A2, 2B1 and 2E1, human 1A1, 1A2, 2A6, 1B1, 3A4 and 2E1, mouse 1B1, and scup fish 1A1 expressed in Chinese hamster V79 cells has been investigated to clarify the role of individual enzymes in the regioselective oxidation of B[c]Ch and the species dependency. In six cell lines expressing individual CYP enzymes from four different species B[c]Ch was metabolized to several isomeric phenols and trans?dihydrodiols. However, cell lines expressing human 3A4, 2A6 and 2E1 or rat 1A2, 2B1 and 2E1 were metabolically in-competent towards B[c]Ch. Among the trans?dihydrodiols the 9,10-isomer could be detected in cells expressing human, rat and fish CYP 1A1 and to a minor extent in cells with human 1A2, but not in cells expressing human and mouse CYP 1B1. The latter two cell lines produced high amounts of the bay region 3,4-dihydrodiol, whereas the K-region 7,8-dihydrodiol was a minor metabolite. Oxidation of B[c]Ch to the 1,2-dihydrodiol could not be catalyzed by any of the CYP enzymes investigated except fish 1A1. Our results suggest that metabolic activation of B[c]Ch is initiated predominantly by CYP 1A1 to result selectively in the formation of fjord region 9,10-dihydrodiol 11,12-epoxides regardless of the species involved. The activation of B[c]Ch appears to be limited by a low regioselectivity for the 9,10-oxidation.     

COMPARATIVE METABOLISM OF PHENANTHRO[3,4-B]THIOPHENE,PHENANTHRO[4,3-B]THIOPHENE AND THEIR CARBON ANALOG BENZO[C]PHENANTHRENE BY RAT LIVER MICROSOMES

Phenanthro[3,4-b]thiophene (P[3,4-b]T) and phenanthro[4,3-b]thiophene (P[4,3-b]T) are thiasters of weakly mutagenic benzo[c]phenanthrene (B[c]P). These polycyclic sulfur heterocycles (thia-PAHs) represent a group of chemicals which have been identified in cigarette smoke. P[3,4-b]T is a potent mutagen in Salmonella typhimurium strain TA100 in the presence of rat liver S9, whereas its isosteric isomer P[4,3-b]T is a nonmutagenic compound. In order to understand the mechanism underlying the differences in the mutagenic activity of P[3,4-b]T and P[4,3-b]T, we have investigated the metabolism of P[3,4-b]T, P[4,3-b]T, and their carbon analogue B[c]P by rat liver microsomes. The liver microsomes from rats treated with Aroclor 1254 metabolized P[3,4-b]T, P[3,4-b]T, and B[c]P at a rate nearly 7- to 9-fold greater than of the control liver microsomes. High-performance liquid chromatography (HPLC) analysis of the metabolites formed showed that B[c]P was metabolized almost exclusively to its dihydrodiols which comprised predominantly K-region diol as noted in the previous studies. Our preliminary studies on the metabolism of P[3,4-b]T, P[4,3-b]T and B[c]P by liver microsomes from control and Aroclor 1254-treated rats have shown a significant reduction in the formation of 6,7-diol (K-region diol) and 8,9-diol (diol with a bay-region double bond) of the two thia-PAHs compared to the formation of analogous 5,6-diol (K-region diol) and 3,4-diol (diol with a bay-region double bond) from B[c]P. Both P[3,4-b]T and P[4,3b]T produced a major, relatively nonpolar metabolite(s) (80–96% of total metabolites). These studies indicate that the highly mutagenic P[3,4-b]T is not metabolized to dihydrodiol with a bay-region double bond to any greater extent than the weakly or nonmutagenic B[c]P or P[4,3-b]T, suggesting that the metabolite(s) other than P[3,4-b]T8,9-diol is likely to be involved in the mutagenicity of P[3,4-b]T.     

Two-component,one-pot synthesis of pyrimido[1,2-b] [1,2,4,5]tetrazines

Derivatives containing the cyclohepta4′,5′thieno[2′,3′:4,5]pyrimido[1,2-b][1,2,4,5]-tetrazin-6-one system were prepared from the reaction of 3-amino-2-thioxo-1,2,3,5,6,7,8,9-octahydro-4H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-one (5) or its 2-methylthio derivative 6 with hydrazonoyl chlorides 9. The mechanism of the studied reactions has been discussed and the biological activity of the isolated products has been evaluated.     

An Abbreviated Synthesis of 7,12-Dimethylbenz[ a ]anthracene and Benzo[ c ]chrysene Metabolites Using the Suzuki Reaction

Efficient syntheses of important metabolites of 7,12-dimethylbenz[ a ]anthracene (DMBA) and benzo[ c ]chrysene (B[ c ]C), via Suzuki coupling reaction are described. This approach provides an excellent method for the preparation of 3-methoxy-DMBA 5 , 10-methoxy-B[ c ]C 14 and 2-methoxy B[ c ]C 20 , and hence for the corresponding dihydrodiols 6 , 15 , and 21 . Following a similar Suzuki reaction, DMBA-6(5 H )-one 8 was also synthesized in high yield.     

11H-Benzo[4,5]imidazo[1,2-a]indol-11-one as a New Precursor of Azomethine Ylides: 1,3-Dipolar Cycloaddition Reactions with Cyclopropenes and Maleimides

The possibility of generating azomethine ylides from 11H-benzo[4,5]imidazo[1,2-a]indol-11-one and amino acids is shown for the first time. Based on the cycloaddition reactions of these azomethine ylides with cyclopropenes and maleimides, cyclopropa[a]pyrrolizines, 3-azabicyclo[3.1.0]hexanes, and pyrrolo[3,4-a]pyrrolizines spiro-fused with a benzo[4,5]imidazo[1,2-a]indole fragment were synthesized. Spirocyclic compounds were obtained in moderate to good yields, albeit with poor diastereoselectivity. Density functional theory calculations were performed to obtain an insight into the mechanism of the 1,3-dipolar cycloaddition of 11H-benzo[4,5]imidazo[1,2-a]indol-11-one-derived azomethine ylides to cyclopropenes. The cytotoxic activity of some of the obtained cycloadducts against the human erythroleukemia (K562) cell line was evaluated in vitro by MTS-assay.     

高效液相色谱法检测植物油中苯并[a]芘的含量

建立并验证高效液相色谱法检测植物油中苯并[a]芘的含量.将植物油样品溶于正己烷中混匀,用苯并[a]芘固相萃取柱净化,用正己烷洗脱苯并[a]芘,荧光检测器检测.苯并[a]芘在0.1～100μg/kg浓度范围内线性相关系数r2=0.9999,本方法平均回收率为96.95%～101.30%,相对标准偏差RsD为0.980%～...     

Mutational Consequences on Replication of M13 Constructs Containing Isomeric Diol Epoxide Adducts in E. coli

Eight isomeric dGuo and eight dAdo adducts resulting from cis and trans opening of each of the four optically active diol epoxides (DEs) derived from benzo[ a ]pyrene (B a P) and benzo[ c ]phenanthrene (B c Ph) were placed in each of two 16-mer DNA sequences to give 32 modified oligonucleotides, which were ligated into M13mp7L2 and allowed to replicate in SOS-induced Escherichia coli . The effects of parent hydrocarbon, adduct stereochemistry, and sequence context on mutagenic response are highly interdependent. B a P DE adducts are generally more mutagenic than the corresponding B c Ph adducts. The mutational frequency is generally larger for cis - relative to trans -opened DE adducts of both dGuo and dAdo. In a ～TA*G～ context, B c Ph DE dAdo adducts (A*) with R configuration at the site of attachment to the adenine base produced very few substitution mutations when compared with adducts having S configuration. This configurational effect is not observed for B a P DE dAdo adducts, nor for B a P or B c Ph dGuo adducts.     

A Coupled-Column HPLC Method for Routine Analysis of Various Chrysene and Benzo[a]Pyrene Metabolites in Urine

The carcinogenic potential of some PAHs leads to the necessity of expressive biological monitoring for people occupationally exposed to PAH. A highly automated, coupled-column, high performance liquid chromatography method for the simultaneous quantification of several chrysene and benzo[a]pyrene metabolites in urine of exposed subjects is presented. After enzymatic hydrolysis of the metabolites, the sample can be directly injected into the HPLC system. The instrumental set-up comprises an analytical column and a precolumn, connected via a 6-port switching valve. Clean-up and analyte preconcentration are automatically performed on the precolumn which is packed with copper-phthalocyanine modified silica. Afterwards, analytes are automatically transferred onto the analytical column where separation is carried out by elution with a methanol-water gradient. Detection of the analytes makes use of their natural fluorescence. Thus, clean-up and analytical separation require little time, making the method suitable for routine analysis in biomonitoring.     

The Role of Cytochrome P450 1B1 in Dibenzo[ a,l ]pyrene-induced Carcinogenesis

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from these animals provided further evidence for the central role of P450 1B1-catalyzed formation of ( m )- anti -DB[ a,l ]PDE-DNA adducts in DB[ a,l ]P-induced carcinogenesis.     

Metabolism Of Benzo[a]Pyrene in Fish Hepatocytes Cultured on Microplates

The metabolism of benzo[a]pyrene (BP) by trout and catfish hepatocytes seeded on 96-well microplates was examined. The 9,10-; 4,5-; and 7,8-dihydrodiols, 1,6-; 3,6- quinones and 3-OH BP were found to be the major metabolites in both the trout and catfish hepatocytes exposed to BP for 4 h. The results were compared with the corresponding long term culture of hepatocytes on porous membranes and in microsomes isolated from the whole liver. It was found that cells cultured on microplates retained their BP metabolizing activities for at least 6 d after seeding. This method has the major advantage that only a small number of cells are required and in parallel, it is possible to perform cytotoxicity and cytogenetic microassays on cell populations characterized for their metabolic capacity.     

Effect of Structure and Sequence on Mutations Induced by Diol Epoxide-DNA Adducts in an E. Coli−M13 Vector System

The effects of two DNA sequence contexts on the mutagenic response to dG and dA adducts derived from the optically active 7,8-diol 9,10-epoxides of benzo[a]pyrene (BaP DEs) and 3,4-diol 1,2-epoxides of benzo[c]phenanthrene (BcPh DEs) were examined. On replication of the adduct-containing, single-stranded vector M13mp 7L2 in E. coli SMH77, frequencies of substitution mutations ranging from 0.05% to 68% were observed. In general, the highest mutational frequencies were observed for BaP DE-dA adducts at the central position in a ～TAG～ sequence. The BaP DE adducts showed no relationship between stereochemistry and mutagenic response. In contrast, mutagenicity of the BcPh DE-dA adducts in the ～TAG～ sequence depended strongly on the configuration at C-1, the site of attachment of the hydrocarbon to the purine. In this sequence, BcPh DE-dA adducts with 1R configuration were 16-600 times less mutagenic than those with 1S configuration.