Mutagenic potential of benzo[a]pyrene-derived DNA adducts positioned in codon 273 of the human P53 gene

Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions. When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.     

Mutational specificity of benzo[a]pyrene diolepoxide in monkey cells

Benzo[a]pyrene diolepoxide (BPDE) is thought to be the major mutagenic and carcinogenic intermediate in benzo[a]pyrene metabolism in mammalian cells. In order to test the mutagenic specificity of this compound in mammalian cells, we have used the pZ189 shuttle vector system to identify and analyze point mutations induced when DNA treated in vitro with BPDE is replicated in monkey cells. We find that point mutations occur almost exclusively at G.C base pairs; G.C----T.A and G.C----C.G transversions and single base pair deletions occur most frequently. This pattern is consistent with the known preferential covalent binding of BPDE to G residues.     

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.     

Depurination of benzo[a]pyrene-diolepoxide treated DNA

Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene. On incubation of the reacted DNA, apurinic sites developed which gave rise to strand breakage in alkaline solution. The reduction in molecular weight produced by these breaks was measured by analytical ultracentrifugation. In the case of anti-BPDE this depurination was shown to occur in two stages. The first was mainly due to attack at the 7-position of guanine, to yield an adduct which was lost from the DNA within a few hours. The second stage was due to much slower loss of the major N2-guanine adduct. The separated enantiomers, (+)- and (-)-anti-BPDE, and syn-BPDE all caused depurination to various extents. It is argued that although these processes are important in a study of the action of BPDE on DNA in vitro, their contribution to the biological activity of BPDE is probably negligible.     

Genotypic mutation analysis in the p53 gene of benzo[a]pyrene-treated European flounder (Platichthys flesus)

We have applied a genotypic mutation detection system (the Restriction Site Mutation (RSM) assay) to detect mutations in the marine teleost flounder (Platichthys flesus). The aim of this study was to evaluate this species as an environmental indicator of genotoxic exposure. We have used the model genotoxin benzo[a]pyrene (B[a]P) to determine the limits of mutation detection in the p53 gene of flounder liver DNA. This study has revealed two important findings. Firstly, we were able to demonstrate that a polymorphism exists in the TaqI restriction site of exon 8 of the flounder p53 gene at codon 243. This polymorphic allele was present as a heterozygote at a mean frequency of 15%, whereas 85% carried the homozygous wild type sequence. Secondly, we established that B[a]P treatment resulted in specific mutational events at the adenine base of the same TaqI site, contrasting previous reports stating that there was a guanine preference for this chemical in mammalian DNA. This difference in mutation specificity may possibly be accounted for by sequence specific factors or by species differences in metabolic activation and/or DNA repair and are worthy of further study.     

Asbestos catalyzes the formation of the 6-oxobenzo[a]pyrene radical from 6-hydroxybenzo[a]pyrene

Crocidolite asbestos catalyzes the oxidation of 6-hydroxybenzo[a]pyrene, a metabolite of benzo[a]pyrene, to the 6-oxobenzo[a]pyrene radical as determined by electron spin resonance spectroscopy. This may be a mechanism whereby inhaled asbestos enhances the incidence of lung cancer induced by cigarette smoke, which contains benzo[a]pyrene.     

Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.     

Oxidation of benzo[a]pyrene by the filamentous fungus Cunninghamella elegans.

Cunninghamella elegans oxidized benzo[a]pyrene to several metabolic products. Compounds that were isolated and identified were: trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene 1,6-quinone, benzo[a]pyrene 3,6-quinone, 9-hydroxybenz[a]pyrene, and 3-hydroxybenzo[a]pyrene. In addition, an unidentified dihydroxybenzo[a]pyrene metabolite was also formed. Experiments with [14C]benzo[a]pyrene showed that over a 96-h period, 18.4% of the hydrocarbon was converted to metabolic products. Most of the metabolites were sulfate conjugates as demonstrated by the formation of benzo[a]pyrene quinones and phenols after treatment with aryl sulfatase. Glucuronide and sulfate conjugates were also detected as water-soluble metabolites. The results show that benzo[a]pyrene is metabolized by a filamentous fungus in a manner that is remarkably similar to that observed in higher organisms.     

Rapid mineralization of benzo[a]pyrene by a microbial consortium growing on diesel fuel

A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-(14)C]benzo[a]pyrene to (14)CO(2) when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations. Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter(-1) and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter(-1). A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds. The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the class Proteobacteria but that the consortium also included members which were related to the genera Mycobacterium and Sphingobacterium.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro

Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor beta and gamma loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system.     

Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system

A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.     

Purification and characterization of a benzo[a]pyrene hydroxylase from Pleurotus pulmonarius

Cytochrome P450 has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by white-rot fungi. We have purified and reconstituted a benzo[a]pyrene hydroxylating cytochrome P450 (P450) from microsomal fractions of the white rot fungus Pleurotus pulmonarius. The microsomal P450 was recovered using a combination of n-aminooctyl agarose and hydroxyapatite chromatography and had an apparent molecular mass of 55 kDa. The purified protein exhibited moderate affinity for benzo[a]pyrene with a K(s) of 66 microM calculated from the Type I substrate binding spectra produced. Reconstitution of activity was achieved and a turnover of 0.75 nmol 3-hydroxybenzo[a]pyrene product/min/nmol P450 was observed, comparable to levels of metabolism observed by animal cytochromes P450 involved in xenobiotic detoxification.     

Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by Aroclor 1254 or phenobarbital

The constitutive and Aroclor 1254-induced activities of hepatic microsomal benzo[a]pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11-day-old noninduced male rats, benzo[a]pyrenediones and 9-hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21-day-old males benzo[a]pyrene-diones and benzo[a]pyrene-9,10-dihydrodiol were predominant. In 60- and 120-day-old animals 3-hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5-dihydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21-day-old immature male rats, in which there was a 330- and 4.5-fold increase in the formation of 3-hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3- to 10.1-fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 mumol/kg) or Aroclor 1254 (100 mumol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120-day-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential induction of sister-chromatid exchanges by benzo[a]pyrene in variant mouse hepatoma cells

Two variant mouse hepatoma cell lines had been separated from a parent cell line, Hepa-1c1c7, by fluorescence activated cell sorting. Earlier metabolic studies had shown that variant TAOc1BPrc1 was more active in the metabolism of the indirect carcinogen benzo[a]pyrene than was variant BPrc1. In an extension of these studies, the relationship between the metabolic capabilities of these two cell lines and the induction of sister-chromatid exchanges by B[a]P was investigated. It was observed that TAOc1BPrc1 yielded a significant dose-dependent increase in the induction of SCE by B[a]P whereas BPrc1 did not show a response significantly greater than control. Metabolic results indicated that the induction of SCE in TAOc1BPrc1 was due to the production of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by this variant. This metabolite did not appear to be produced by BPrc1. Furthermore, TAOc1BPrc1 required only 40 nM B[a]P to induce a 2-fold increase in SCE frequency. This concentration is considerably lower than that required to elicit a similar response in other reported cell lines. To our knowledge, this is the first report of the use of a mouse hepatoma cell line for determining the relationship of metabolic capability to the induction of SCE.     

Excretion of benzo[a]pyrene-Gua adduct in the urine of benzo[a]pyrene-treated rats

A benzo[a]pyrene(BP)-Gua adduct was extracted in the urine of rats treated with BP. Some (0.15%) of the administered dose of BP was excreted as BP-Gua within 48 h. A double labelling experiment demonstrated that the excreted product contained both a BP and a Gua moiety. Partially hepatectomized rats treated with [14C]Gua during the regenerative phase were injected with [3H]BP and the urine collected and processed by chromatographic procedures. The adduct had similar chromatographic properties to the adduct released from human PLC/5 cells treated with 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and co-chromatographed with 7-BPDE-Gua released from BPDE-adducted DNA under aqueous conditions. Detection and quantitation of BP-Gua offers an alternative, non-invasive method of monitoring individuals exposed to carcinogenic polycyclic aromatic hydrocarbons (PAHs).     

Origins of stereospecificity in DNA damage by anti-benzo[a]pyrene diol-epoxides. A molecular modelling study

A general computational procedure for the modelling of intercalated DNA-ligand complexes has been developed, and is used here to model intercalated complexes of the (+)-anti and (-)-anti enantiomers of benzo[a]pyrene diol-epoxide (BPDE) with cytosine-3',5'-guanosine double-stranded DNA sequences (dCpG). Results are presented indicating differences between the behaviours of the two enantiomers which have implications for the understanding of the stereospecificity of DNA strand breakage by benzo[a]pyrene diol-epoxides.     

The Extreme Variety of Genotoxic Response to Benzo[a]pyrene in Three Different Human Cell Lines from Three Different Organs

Polycyclic aromatic hydrocarbons (PAHs) are associated with occupational exposure and urban atmospheric pollution. Determination of the genotoxic properties of these compounds is thus of outmost importance. For this purpose a variety of cellular models have been widely used. Reliable results can however only be obtained with models reflecting the specific sensitivity of different organs towards PAHs. In this work, we compared the response to benzo[a]pyrene in cell lines from human lungs (A549) and bladder (T24); two important target organs for PAHs-induced cancer. Human hepatocytes (HepG2) were used as a reference, although liver is not a concern for PAHs carcinogenesis. Adducts arising from the ultimate diol-epoxide metabolite of B[a]P, BPDE, were found to be produced in a dose-dependent manner in HepG2. BPDE DNA adducts were not detected in T24 and in A549 their formation was found to be most efficient at the lowest concentration studied (0.2 µM). These results are probably explained by differences in induction and activity of phase I metabolization enzymes, as well as by proteins eliminating the B[a]P epoxide in A549. In addition to BPDE adducts, oxidative DNA damage, namely strand breaks and oxidized purines were measured and found to be produced only in minute amounts in all three cell lines. In summary, our results emphasize the large differences in the response of cells originating from different organs. Our data also point out the importance of carefully selecting the doses used in in vitro toxicological experiments. The example of A549 shows that working at high doses may lead to an underestimation of the risk. Finally, the choice of method for evaluating genotoxicity appears to be of crucial importance. The comet assay does not seem to be the best method for a compound like B[a]P which induces stable adducts causing limited oxidative damage.