Analysis of tetrahydroxylated benzo[a]pyrene isomers in hair as biomarkers of exposure to benzo[a]pyrene.

A first gas chromatography-tandem mass spectrometry (GC-MS/MS) method was designed for analysis of four tetrahydroxylated benzo[a]pyrene metabolites (benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol, benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol, benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol, and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol) in hair. Hair powder extract was submitted to liquid-solid extraction, followed by C18 solid-phase purification. The analytes were derivatized with use of N-methyl-N-(trimethylsilyl)trifluoroacetamide and then analyzed by GC-MS/MS in negative chemical ionization mode. The calibration curve was linear from the limit of quantification (LOQ) to 20 pg/mg in hair. The coefficient of determination of the calibration curve was more than 0.975 for all the analytes investigated. The LOQs ranged from 0.075 to 0.2 pg/mg in hair. The method was afterward applied to the analysis of hair of 16 rats randomly allocated to experimental groups receiving 16 polycyclic aromatic hydrocarbons solubilized in oil at 0 or 0.8 mg/kg body weight by oral administration three times per week for 90 days. The analysis of monohydroxylated and dihydroxylated benzo[a]pyrenes was conducted in parallel by GC-MS/MS on the same samples. All tetrahydroxylated benzo[a]pyrene isomers were detected in hair samples collected from rats exposed to polycyclic aromatic hydrocarbons. Benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol, the most abundant isomer in hair of treated rats, was also the principal isomer released in DNA adduct hydrolysis in humans. Moreover, the benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol concentrations in hair were significantly greater than those of 2-hydroxybenzo[a]pyrene, 1-hydroxybenzo[a]pyrene, 7-hydroxybenzo[a]pyrene, and 4-hydroxybenzo[a]pyrene and similar to those of 9-hydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene. The method was also sufficiently sensitive to monitor environmental levels of exposure because two hair specimens in the eight analyzed from smokers were above the LOQ for benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. This study therefore demonstrated that tetrahydroxylated benzo[a]pyrenes in hair might be a useful biomarker for the assessment of both the general population and occupationally exposed workers.

In vivo examination of the genotoxicity of the urban air and surface soil pollutant, 3,6-dinitrobenzo[e]pyrene, with intraperitoneal and intratracheal administration.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP) was identified as a new potent mutagen toward Salmonella strains in surface soil and airborne particles. Because data of in vivo examination of the genotoxicity of 3,6-DNBeP are limited, micronucleus test was performed in peripheral blood and bone marrow, and comet assay in the lungs of mice treated with 3,6-DNBeP. In male ICR mice intraperitoneally (i.p.) injected with 3,6-DNBeP, the frequency of micronuclated polychromatic erythrocytes (MNPCEs) was increased in the peripheral blood and bone marrow after 24 h in a dose-dependent manner. Compared to controls, the highest dose of 3,6-DNBeP (40 mg/kg B.W.) induced 7.3- and 8.7-fold increases of MNPCE frequency in the peripheral blood and bone marrow, respectively. Furthermore, when 3,6-DNBeP was intratracheally (i.t.) instilled to male ICR mice, 3,6-DNBeP at the highest dose of 0.1 mg/kg body exhibited 3.1-fold increase of DNA tail moment in the lungs at 3 h after the instillation compared to controls. The values of DNA tail moment at 9 and 24 h after the instillation were increased up to 3.5 and 4.2-fold, respectively. These data indicate that 3,6-DNBeP is genotoxic to mammalians in in vivo and suggest that 3,6-DNBeP may be a carcinogenic compound present in the human environment.

ERCC1 and ERCC2 haplotype modulates induced BPDE-DNA adducts in primary cultured lymphocytes.

BACKGROUND: Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair (NER), of which ERCC1 and ERCC2/XPD exert an indispensable role. Genetic variations in ERCC1 and ERCC2 have been related to DNA repair efficiency. In this study we used lymphocytes from healthy individuals to show that polymorphisms in ERCC1 and ERCC2 are directly associated with decreased DNA repair efficiency. METHODS: ERCC1 (rs3212986 and rs11615) and ERCC2 (rs13181, rs1799793 and rs238406) were genotyped in 818 healthy Han individuals from the northeast of China. BPDE induced DNA adducts in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 282 randomly selected participants. The effect of ERCC1 rs3212986 and ERCC2 rs238406 on DNA damage caused by B[a]P was assessed with a modified comet assay. RESULTS: We found that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 were associated with the high levels of BPDE-DNA adducts. Especially ERCC1 rs3212986 A-allele variant was significantly associated with the high BPDE-DNA adducts. Haplotype analysis showed that the ERCC1 haplotype AC (OR = 2.36, 95% CI = 1.84-2.97), ERCC2 haplotype AGA (OR = 1.51, 95% CI = 1.06-2.15) and haplotype block AGAAC (OR = 5.28, 95% CI = 2.95-9.43), AGCAC (OR = 1.35 95% CI = 1.13-1.60) were linked with high BPDE-DNA adducts. In addition, we found that the combined minor alleles of ERCC1 rs3212986 and ERCC2 rs238406 were associated with a reduced DNA repair capacity. CONCLUSIONS: Our results suggest that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 are associated with decreased repair efficiency of BPDE induced DNA damage, and may be predictive for an individual's DNA repair capacity in response to environmental carcinogens.

Screening and characterization of new monoclonal anti-benzo[a]pyrene antibodies using automated flow-through microarray technology.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10 ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect on humans. In the present investigation, mice were immunized with B[a]P-bovine serum albumin conjugates and 110 generated hybridoma cell lines screened by different techniques to identify clones that produce anti-B[a]P antibodies. Subsequently, a new automated flow-through biochip noncompetitive direct chemiluminescence immunoassay (CLEIA) was compared with conventional indirect and direct enzyme-linked immunosorbent assays (ELISAs). It was demonstrated that the microchip-based screening method compared to ELISA was fast and very sensitive with use of only nanoliter volumes of supernatant. Forty clones could be evaluated in less than 5 min. Six high affinity monoclonal antibodies with different cross-reactivities (CR) for individual PAHs were identified by the chip-based assay and indirect microtiter plate ELISA. In comparison, the direct ELISA in the microtiter plate failed to identify three of these clones. The four antibodies with the highest affinity had half maximum inhibitory concentrations (IC(50) values) between 0.31 and 0.92 µg/L for B[a]P. Affinity constants of these four antibodies were determined by surface plasmon resonance using a water soluble B[a]P-peptide. The observed CR pattern of the four monoclonal antibodies for 16 tested PAHs was quite different. Only one specific antibody for B[a]P was observed, while others were more suitable for class-specific PAH determination.

Characterisation of B(a)P metabolites formed in an ex vivo pig skin model using three complementary analytical methods.

An original method was developed to separate, identify and quantify the different benzo(a)pyrene (B(a)P) metabolites formed through oxidative and conjugative pathways. All B(a)P metabolites were separated by an improved high-performance liquid chromatography method, then detected and quantified relatively by online radioactivity detection. At the same time, metabolite structures were characterised by tandem mass spectrometry using two complementary ionisation modes: electrospray ionisation in the negative mode and atmospheric pressure chemical ionisation in the positive mode. This method was successfully applied to the analysis of B(a)P metabolites, produced by incubation of B(a)P with the ex vivo pig ear skin model. These include glucuronic acid and sulphate conjugates of B(a)P-OHs and B(a)P-diols, as well as direct phase I metabolites: B(a)P-tetrol, B(a)P-diones, B(a)P-catechols, B(a)P-diols and B(a)P-OHs.

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines.

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.

The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MS.

Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (+/-)-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [(+/-)-anti-benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5'-ACCCG5CG7TCCG11CG13C-3'/5'-GCGCGGGCGCGGGT-3') derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational 'hot spots' and have also been reported as chemical 'hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5 >> G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5 >> G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G(7)) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run.

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimurium umu strains expressing human cytochrome P450 and N-acetyltransferase.

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.

Genotoxicity of 3,6-dinitrobenzo[e]pyrene, a novel mutagen in ambient air and surface soil, in mammalian cells in vitro and in vivo.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 10(5) clonable cells, which increased to 229 per 10(5) clonable cells after treatment with 1.0 microg/ml (3 microM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 10(5) clonable cells by treatment of 1.0 microg/ml (3 microM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 microg/ml (3 microM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.

The role of electronic properties to the mutagenic activity of 1,6- and 3,6-dinitrobenzo[a]pyrene isomers.

Equilibrium geometries, infrared spectra, vertical first ionization potential (IP), electronic affinity (EA), dipole moment (mu) and electronic dipole polarizability (alpha) of 1,6- and 3,6-dinitrobenzo[a]pyrene isomers (1,6-DNBaP and 3,6-DNBaP) were evaluated by means of Density Functional Theory (DFT) and recent semiempirical PM6 method. Structural, energetic and vibrational properties of DNBaP isomers are substantially similar to each other. Calculated IP, EA and alpha values of these isomers are practically identical, while mu of 3,6-DNBaP (8.2 D at DFT level) is predicted to be ca. 4 times the value of 1,6-DNBaP isomer (1.9 D at DFT level), owing to favorable mutual orientation of the individual nitro group vectors. Higher direct-mutagenic activities of 3,6-DNBaP with respect to 1,6-DNBaP isomer by 1-2 orders of magnitude might be determined by its peculiar electronic charge distribution, which through stronger electrostatic and inductive interactions, can promote much more effectively binding to active-site of enzymes involved in mutagenic pathways. On the other hand, orientation of the nitro substituents relatively to the plane of the aromatic moiety, molecular sizes, as well as nitroreduction and oxidation reactions seem not to have a key role in the determination of the different mutagenic behaviour of these isomers.

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens.

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.

Oxidized metabolites from benzo[a]pyrene, benzo[e]pyrene, and aza-benzo[a]pyrenes. A computational study of their carbocations formed by epoxide ring opening reactions.

A DFT study aimed at understanding structure-reactivity relationships and fluorine substitution effects on carbocation stability in benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), and aza-benzo[a]pyrene (aza-BaP) derivatives are reported. The relative energies of the resulting carbocations are examined and compared, taking into account the available biological activity data on these compounds. O-Protonation of the epoxides and diol epoxides leads to carbocation formation by barrierless processes. Charge delocalization modes in the resulting carbocations were deduced via NPA-derived changes in charges, and fluorine substitution effects were analyzed on the basis of charge density at different carbocation positions. Thus, fluorine substitution at sites bearing negative charge generated inductive destabilization of the carbocation, whereas a fluorine atom at a ring position which presented significant positive charge density produced a less pronounced destabilization due to fluorine p-pi back-bonding. Protonation reactions were also studied for the azaBaPs. In selected cases, the covalent adducts generated via bond formation with the exocyclic nitrogen of cytosine were computed and relative energies and geometries of the resulting adducts were examined.

Determination of 3,6-dinitrobenzo[e]pyrene in surface soil and airborne particles by high-performance liquid chromatography with fluorescence detection.

We developed a sensitive analytical method and an efficient clean-up method to quantify 3,6-dinitrobenzo[e]pyrene (3,6-DNBeP) in surface soil and airborne particles. After purification using a silica gel column and two reversed-phase columns, 3,6-DNBeP was reduced to 3,6-diaminobenzo[e]pyrene by a catalyst column and analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector. 3,6-DNBeP was detected in all of the soil samples and airborne particles examined. The concentration of 3,6-DNBeP in surface soil and airborne particles was determined in the ranges of 347-5007 pg/g of soil and 137-1238 fg/m3, respectively.

Synthesis and cytotoxic activity of benzo[a]pyrano[3,2-h] and [2,3-i]xanthone analogues of psorospermine, acronycine, and benzo[a]acronycine.

Condensation of 2-hydroxy-1-naphthalenecarboxylic acid with phloroglucinol afforded 9,11-dihydroxy-12H-benzo[a]xanthen-12-one (6). Construction of an additional dimethylpyran ring onto this skeleton, by alkylation with 3-chloro-3-methyl-1-butyne followed by Claisen rearrangement, gave access to 6-hydroxy-3,3-dimethyl-3H,7H-benzo[a]pyrano[3,2-h]xanthen-7-one (12) and 5-hydroxy-2,2-dimethyl-2H,6H-benzo[a]pyrano[2,3-i]xanthen-6-one (13), which were methylated into 6-methoxy-3,3-dimethyl-3H,7H-benzo[a]pyrano[3,2-h]xanthen-7-one (14) and 5-methoxy-2,2-dimethyl-2H,6H-benzo[a]pyrano[2,3-i]xanthen-6-one (15), respectively. Osmium tetroxide oxidation of 14 and 15 gave the corresponding (+/-)-cis-diols 16 and 17, which afforded the corresponding esters 18-21 upon acylation. Similarly, condensation of 2-hydroxy-1-naphthalenecarboxylic acid with 3,5-dimethoxyaniline gave 11-amino-9-methoxy-12H-benzo[a]xanthen-12-one (23) which was converted into 11-amino-9-hydroxy-12H-benzo[a]xanthen-12-one (24) upon treatment with hydrogen bromide in acetic acid. Alkylation with 3-chloro-3-methyl-1-butyne followed by Claisen rearrangement afforded 6-amino-3,3-dimethyl-3H,7H-benzo[a]pyrano[3,2-h]xanthen-7-one (25) and 5-amino-2,2-dimethyl-2H,6H-benzo[a]pyrano[2,3-i]xanthen-6-one (26). The new benzopyranoxanthone derivatives only displayed marginal antiproliferative activity when tested against L1210 and KB-3-1 cell lines. The only compounds found significantly active against L1210 cell line, 16 and 20, belong to the benzo[a]pyrano[3,2-h]xanthen-7-one series, which possess a pyran ring fused angularly onto the xanthone basic core.

Benzo[a]pyrene-7,8-quinone-3'-mononucleotide adduct standards for 32P postlabeling analyses: detection of benzo[a]pyrene-7,8-quinone-calf thymus DNA adducts.

Benzo[a]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). The formation of BPQ from B[a]P through trans-7,8-dihydroxy-7,8-dihydroB[a]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[a]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species. We previously reported the complete characterization of four novel stable BPQ-deoxyguanosine (dG) and two BPQ-deoxyadenosine (dA) adducts (Balu et al., Chem. Res. Toxicol. 17 (2004) 827-838). However, the identification of BPQ-DNA adducts by 32P postlabeling methods from in vitro and in vivo exposures required 3'-monophosphate derivatives of BPQ-dG, BPQ-dA, and BPQ-deoxycytidine (dC) as standards. Therefore, in the current study, BPQ adducts of dGMP(3'), dAMP(3'), and dCMP(3') were prepared. The syntheses of the BPQ-3'-mononucleotide standards were carried out in a manner similar to that reported previously for the nucleoside analogs. Reaction products were characterized by UV, LC/MS analyses, and one- and two-dimensional NMR techniques. The spectral studies indicated that all adducts existed as diastereomeric mixtures. Furthermore, the structural identities of the novel BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adducts were confirmed by acid phosphatase dephosphorylation of the BPQ-nucleotide adducts to the corresponding known BPQ-nucleoside adduct standards. The BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adduct standards were used in 32P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32P postlabeling analysis revealed the formation of 8 major and at least 10 minor calf thymus DNA adducts. Of these BPQ-DNA adducts, the following were identified: 1 BPQ-dGMP adduct, 2 BPQ-dAMP adducts, and 3 BPQ-dCMP adducts. This study represents the first reported example of the characterization of stable BPQ-DNA adducts in isolated mammalian DNA and is expected to contribute significantly to the future BPQ-DNA adduct studies in vivo and thereby to the contribution of BPQ in B[a]P carcinogenesis.

Epimerization and stability of two new cis-benzo[a]pyrene tetrols by the use of liquid chromatography-fluorescence and mass spectrometry.

Quantitative determination of the hydrolysis products from proteins and DNA gives valuable information regarding the reactive metabolite that forms the protein and DNA adduct. Quantification of protein-benzo[a]pyrene (BP) adducts represents a more sensitive method than quantification of BP-DNA adducts. The aim of the present study was to identify two hydrolysis products from BP-derived protein adducts found in vitro and in vivo in a previous study. Male Wistar rats were injected i.p. with BP, and serum albumin was isolated and subjected to acid hydrolysis at 70 degrees C for 3 h. The hydrolysate was subjected to LC separation, and fractions of the two unknown compounds were collected. The molecular masses of the two unknown compounds were in accordance with being tetrols as judged by LC electrospray mass spectrometry. The fragmentation patterns were characteristic of tetrols with formation of the molecular ion and the loss of water molecules. In addition, the compounds were subjected to acid hydrolysis at 70 degrees C with 0.1 M HCl for 3 h. We observed that two of the known tetrols epimerized to the two unknown tetrols and vice versa. This is probably a characteristic epimerization involving not only position C(10)-OH but also another site like position C(7)-OH. The in vivo findings of the two unknown adducts are probably the result of the formation of BPDE III in the metabolism of BP. These two tetrols must then have the C(7)-OH and C(8)-OH groups in a cis position.

Flow-through partial-filling affinity capillary electrophoresis using a crossreactive antibody for enantiomeric separations.

It is demonstrated that the separation of diastereoisomers and enantiomers can be accomplished by the flow-through partial-filling affinity CE using a crossreactive mAb. This approach revealed differences in the binding strength of the (+/-)-cis- and (+/-)-trans-benzo[a]pyrene tetrols with the anti-polycyclic aromatic hydrocarbon mAb and demonstrated that (+)-enantiomers are more strongly immunocomplexed than their (-)-counterparts. It is proposed that crossreactive monoclonal antibodies (i.e. mAb raised against achiral molecule and possessing limited selectivity) could be effectively utilized for specific stereoisomeric differentiation and chiral separations.

Detection of a novel mutagen, 3,6-dinitrobenzo[e]pyrene, as a major contaminant in surface soil in Osaka and Aichi Prefectures, Japan.

We previously identified 1,3-, 1,6-, and 1,8-dinitropyrene (DNP) isomers as major mutagens in surface soil in three metropolitan areas of Japan. In the present study, an organic extract from surface soil collected at a park in Takatsuki in Osaka Prefecture, which showed extremely high mutagenicity in Salmonella typhimurium TA98 in the absence of mammalian metabolic system (S9 mix), was investigated to identify major mutagens. A new powerful bacterial mutagen, as well as 1,6- and 1,8-DNP isomers, was isolated from the organic extract (1.8 g) of the soil sample (2.2 kg) by column chromatography. On the basis of mass spectra, the new mutagen, which accounted for 15% of the total mutagenicity of the soil extract, was thought to be a dinitrated polycyclic aromatic hydrocarbon with a molecular weight of m/z 342. The mutagen was synthesized from benzo[e]pyrene by nitration and was determined to be 3,6-dinitrobenzo[e]pyrene (DNBeP) based on its 1H NMR spectrum. The mutagenic potency of 3,6-DNBeP in the Ames/Salmonella assay was extremely high, in that it induced 285,000 revertants/nmol in TA98 and 955,000 revertants/nmol in YG1024 without S9 mix and was comparable to those of DNP isomers, which are some the most potent bacterial mutagens reported so far. In addition to the soil sample from Takatsuki, 3,6-DNBeP was also detected in surface soil samples collected at parks in four different cities, i.e., Izumiotsu and Takaishi in Osaka Prefecture and Nagoya and Hekinan in Aichi Prefecture, and accounted for 22-29% of the total mutagenicity of these soil extracts in TA98 without S9 mix. These results suggest that 3,6-DNBeP is a major mutagen in surface soil and may largely contaminate the surface soil in these two regions in Japan.

Competing roles of cytochrome P450 1A1/1B1 and aldo-keto reductase 1A1 in the metabolic activation of (+/-)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene in human bronchoalveolar cell extracts.

(+/-)-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol), a proximate carcinogen derived from benzo[a]pyrene (BP) requires further metabolic activation to exert its carcinogenic effects. Two principal pathways have been implicated, and these involve either the formation of (+/-)-trans-7,8-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) catalyzed by P450 1A1/P450 1B1 (NADPH-dependent monoxygenases) or the formation of benzo[a]pyrene-7,8-dione (BP-7,8-dione) catalyzed by human aldo-keto reductases AKR1A1 and AKR1C1-AKR1C4 [NAD(P)(H)-dependent oxidoreductases]. The relative contributions of the two pathways to PAH activation are unknown. In this study, BP-7,8-diol metabolism was studied in human bronchoalveolar H358 cell extracts. Parental H358 cells do not constitutively express P450 1A1/P450 1B1 or AKRs but were manipulated by induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to express P450 1A1/P450 1B1 or by stable transfection to express AKR1A1 (aldehyde reductase). TCDD induction of AKR1A1 transfectants provided a cell line that expressed both pathways. Extracts derived from parental H358 cells plus TCDD (P450 induction) produced electrophilic anti-BPDE, which hydrolyzed to benzo[a]pyrene tetrahydrotetrols (BP-tetrols), extracts derived from AKR1A1-transfected cells (AKR1A1 expression) produced reactive and redox-active BP-7,8-dione, which was trapped in situ as its mono(thioether) conjugate, and extracts derived from AKR1A1 transfectants plus TCDD (coexpression of P450 1A1/P450 1B1 and AKR1A1) produced both anti-BPDE and BP-7,8-dione. The competing activation of BP-7,8-diol by P450 1A1/P450 1B1 and AKR1A1 was studied with varied NADPH:NAD+ ratios. The system with a relatively higher concentration of NADPH favored formation of anti-BPDE via P450 1A1/P450 1B1, while the system with the higher concentration of NAD+ favored formation of BP-7,8-dione via AKR1A1. Under conditions that mimic the cellular redox state, 10 microM NADPH and 1 mM NAD+, equal amounts of BP-tetrols and BP-7,8-dione were formed. This suggests that P450 1A1/P450 1B1 and AKR1A1 play competing roles in the metabolic activation of BP-7,8-diol and that the dominant pathway of BP-7,8-diol activation depends on the redox state of the cells. These model systems provide a cellular context in which the dominant DNA adducts/lesions formed by either pathway may be compared.