Benzo[a]pyrene Diol Epoxide Research Articles

Kinetics of the reaction of a potential chemopreventive agent, 2,6-dithiopurine, and its major metabolite, 2,6-dithiouric acid, with multiple classes of electrophilic toxicants.

Purinethiols are a class of potential cancer chemopreventive agents that exhibit nucleophilic scavenging activity against the carcinogenic electrophile benzo[a]pyrene diol epoxide (BPDE). Of the purinethiols tested previously, 2,6-dithiopurine (DTP), exhibited the highest scavenging activity for BPDE when tested either in vitro or in vivo. Sulfur-based nucleophiles are typically classified as "soft" nucleophiles, showing selectivity in nucleophilic substitution reactions for "soft", easily polarizable electrophiles. It was of interest to determine whether electrophilic toxicants other than BPDE react facilely with DTP, and whether 2,6-dithiouric acid (DUA), the major in vivo metabolite of DTP, also has scavenging activity. Four diverse toxicants tested in the present work, acrolein, melphalan, dimethyl sulfate, and cisplatin, all react facilely with DTP in vitro near neutral pH. These toxicants are expected to react as "soft" electrophiles. Furthermore, each of these compounds, as well as BPDE, reacts with DUA with rate constants comparable to the analogous rate constants for reaction with DTP. In contrast, several toxicants classified as "hard" electrophiles (ethyl methanesulfonate, methylnitrosourea, ethylnitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine) show no appreciable reaction with DTP. These results suggest that both DTP and its major metabolite act as "soft" nucleophiles in nucleophilic substitution reactions and may be effective in scavenging a wide range of toxicants that react as "soft" electrophiles.

32P-postlabelling of bulky human DNA adducts enriched by different methods including immunoaffmity chromatography

DNA adducts in lymphocytes and granulocytes of men exposed occupationally and environmentally to high concentrations of aromatic compounds in air were measured by the 32P-postlabelling method. Adducts in the same samples were characterised using nuclease P1 enrichment, butanol extraction and immunoaffinity purification with an antiserum raised against benzo[ a]pyrene diol epoxide (BPDE). Only part of the adducts found in human samples were extracted by butanol. It also seemed, that only a small part of them belonged to the group of polycyclic aromatic hydrocarbons (PAHs) recognised by the antibody. Relative content of hydrophobic adducts and those with a structure similar to PAHs was higher in winter samples (when exposure to aromatic chemicals in air was higher) in comparison to samples collected in summer.

Impact of the stereochemistry of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide-deoxyadenosine adducts on resistance to digestion by phosphodiesterases I and II and translesion synthesis with HIV-1 reverse transcriptase.

Spatial orientations of bulky DNA adducts can influence the extent of resistance to digestion by exonucleases and translesion synthesis by HIV-1 reverse transcriptase (HIV-1 RT). In order to determine how different diastereomers of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE)-adducted DNAs influence the activity of these enzymes, 11-mer and 33-mer oligodeoxyribonucleotides were synthesized bearing site-specific and stereospecific BPDE adducts at adenine N6 on position two of the human N-ras codon 61. Phosphodiesterase I, which hydrolyzes DNA in the 3'-->5' direction, exhibited greater resistance opposite the lesion with C10-R BPDE-adducted templates than the corresponding C10-S adducts. However, the opposite stereoselective resistance to digestion was observed with phosphodiesterase II, which hydrolyzes DNA in the 5'-->3' direction. These results are complemented by the in vitro replication pattern exhibited with HIV-1 RT. Primer extension reactions under conditions defining single encounters between polymerase and substrate revealed adduct-dependent termination one base 3' to each of the lesions. When experimental conditions were altered to permit multiple encounters, HIV-1 RT was able to replicate past the damaged site on four of the six adducted templates, exhibiting little pausing opposite the lesion. Analyses of the replication pattern past these lesions revealed two general categories of replication blockage, which, like the exonucleolytic digestion data, were also based on the C10-R and C10-S configuration of the stereoisomers. Thus, the chirality of BPDE-dA adducts modulates enzymatic functions. Furthermore, the (+)- and (-)-anti-trans-BPDE-dA modified templates exhibited the most facile bypass, while the (+)- and (-)-anti-cis-BPDE adducts were most blocking.

Lack of Correlation between Degree of Interference with Transcription and Rate of Strand Specific Repair in the HPRT Gene of Diploid Human Fibroblasts

The model that transcription-coupled excision repair reflects the interference of DNA damage with the transcription process predicts that the rate of such excision repair will be related to the degree to which a particular type of lesion blocks transcription. We tested this by measuring the rate of excision repair of guanine adducts formed in the HPRT gene of diploid human fibroblasts and in the overall genome by two structurally related polycyclic carcinogens, 1-nitrosopyrene (1-NOP) and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) and comparing the results with those we found previously using benzo[a]pyrene diol epoxide (BPDE). We also measured the degree of interference with in vitro transcription by these adducts. Our results showed that, although BPDE adducts are four times more effective than 1-NOP adducts in blocking transcription, the preferential and strand-specific repair of 1-NOP adducts was twice as fast as that of BPDE adducts. Excision repair of N-AcO-AAF adducts was significantly slower than that of BPDE adducts and was not strand-specific. The efficiency of blocking of transcription by deacetylated N-AcO-AAF adducts was similar to 1-NOP adducts. Therefore, the extent to which a particular lesion blocks transcription in vitro does not predict its rate of preferential or transcription-coupled excision repair.

Benzo(a)pyrenediolepoxide-hemoglobin adducts and 3-hydroxy-benzo(a)pyrene urinary excretion profiles in rats subchronically exposed to benzo(a)pyrene.

The time profiles of benzo(a)pyrenediolepoxide (BaPDE)-hemoglobin (Hb) adduct formation and 3-hydroxybenzo(a)pyrene (3-OHBaP) urinary excretion were studied in male Sprague-Dawley rats exposed to daily benzo(a)pyrene (BaP) intraperitoneal doses of 1.25, 6.25, and 31.25 mumol/kg administered Tuesday to Friday for 4 consecutive weeks. Blood was withdrawn weekly, on Tuesdays, prior to dosing. Twenty-four-hour urine samples were collected on Mondays (following 72 h without treatment) and Thursdays. Analytes were quantified by high performance liquid chromatography (HPLC)/fluorescence. Exposure to BaP resulted in the accumulation of BaPDE-Hb adducts, reaching an average of 1.2 +/- 0.3, 8.3 +/- 1.9, and 38.2 +/- 6.1 pmol/g Hb for the 1.25, 6.25, and 31.25 mumol/kg per day doses after 4 weeks of treatment. The expected saw tooth excretion profile of 3-OHBaP was observed, with peaks on Thursdays and troughs on Mondays, and showed a progressive rise on both Mondays and Thursdays. Increase in Monday values with time suggested a possible increase in BaP body burden during exposure. To verify this aspect further, the urinary excretion kinetic of 3-OHBaP following acute intraperitoneal dosing (31.25 mumol/kg) was determined. Urine samples were collected at frequent timed intervals for up to 164 h post-dosing. Two-step elimination was observed, the second step having a half-life of 25 h, presumably linked to the slow release of BaP accumulated in fatty tissues upon repeated treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in Vitro Replication Consequences of Stereoisomeric Benzo[a]pyrene-7,8-dihydrodiol 9,10-Epoxide Adducts on Adenine N6 at the Second Position of N-ras Codon 61

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.

Preliminary in vitro investigation into the use of alkaline elution assay for the biomonitoring of humans exposed to genotoxic agents.

1. This in vitro study was undertaken as a preliminary approach before assessing whether the alkaline elution assay can be applied to peripheral blood lymphocytes (PBL) for the monitoring of humans exposed to genotoxic agents such as polycyclic aromatic hydrocarbons (PAH). We have compared in vitro, with the aid of the alkaline elution assay, the formation and the repair of DNA single-strand breaks (ssb) induced by different genotoxic agents [gamma-irradiation, ethyl methanesulfonate (EMS), benzo<a>pyrene diol epoxide (BPDE)] on quiescent and PHA-stimulated human lymphocytes and on a fibroblast cell line. 2. Gamma-irradiation (4 Gy) induced an equivalent amount of DNA ssb in the three cell types. On the other hand, after treatment with EMS (10 mM) and BPDE (50 microM), a higher production of DNA ssb was observed in replicating cells (PHA-stimulated lymphocytes and fibroblasts) when compared with quiescent lymphocytes. 3. After gamma-irradiation, all cell types repaired more than 65% of ssb within 1 h. After treatment with EMS, we noted a deficient DNA repair capacity in quiescent lymphocytes in comparison with replicating cells. In all cell types treated with BPDE, more breaks were observed after a 2 h repair period than immediately after treatment, demonstrating the involvement of a slow repair mechanism after BPDE treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide adducts.

DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in approximately 16% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3'-->5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.

Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides.

Antisera developed against benzo[a]pyrene diolepoxide (BPDE)-DNA adducts are sensitive tools for detection of DNA adducts in human samples. All antisera currently used for biomonitoring studies were produced against DNA or guanosine modified with racemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbent assay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565-572, 1994) recently tested polyclonal and monoclonal (5D2) antisera for cross-reactivity against oligonucleotides containing (+)-and (-)-trans-anti-BPDE-N2-guanine or N6-adenine adducts and showed different stereospecificity for the two antisera. Because of the importance of antiserum specificity in human biomonitoring studies, we have tested several monoclonal (Mab 5D11 and 5D2) and polyclonal (Pab #29) antisera developed against racemic anti-BPDE-DNA adducts, and Mab 8E11 developed against anti-BPDE-guanosine adducts. Stereoisomeric anti-BPDE-modified oligonucleotide adducts in the sequence 5'-d(CC-AT-CG*CTACC)-3' where G* = anti-BPDE-N2-dG with (+)-trans, (-)-trans, (+)-cis and (-)-cis adduct stereochemistry at the C10 position of anti-BPDE were tested by competitive ELISA. Two structurally related 5-methylchrysene diolepoxide adducts with G* = (+)- and (-)-trans-anti-5-MeCDE-N2-dG in the same oligonucleotide were also tested. While Mab5D2 had the highest affinity for the (-)-trans-anti-BPDE-modified oligomer, Mab 5D11 and 8E11 and Pab #29 recognized the (+)-trans-anti-BPDE-modified oligomer better than the (-)-trans-anti-BPDE modified oligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modified DNA adducts better than trans-anti-BPDE-modified oligonucleotides; however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNA adducts and (+)- and (-)-trans-anti-BPDE-modified oligomers. All antisera exhibited lower reactivities with both 5-MeCDE modified oligomers. Because of their sensitive detection of (+)-trans-anti-BPDE-dG adducts, the primary adduct produced in vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurement of most adducts formed in humans.

Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[ a]pyrene: Studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen

Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[ a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 μM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase ( K i ∼ 120 μM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.

Single-base deletion induced by benzo[ a]pyrene diol epoxide at the adenine phosphoribosyltransferase locus in human fibrosarcoma cell lines

Benzo[ a]pyrene diol epoxide (BPDE), a metabolic product of benzo[ a]pyrene, is one of the most widely distributed environmental carcinogens. In this study, we demonstrate that BPDE produces a dose-dependent increase in the frequency of APRT gene reversion in the APRT-deficient cell line, HTD114, which contains single nucleotide insertions at different positions in each APRT allele. The highest reversion frequency observed after BPDE exposure was 3.3 ± 0.9 × 10 −5, at least 10 3-fold greater than the spontaneous frequency. Reversion of either mutant allele was observed to be a consequence of a frame-restoring loss of a single nucleotide. A similar frequency of BPDE-induced reversion at APRT also was observed in a cell line containing only one type of the mutant alleles of HTD114, thus eliminating the possibility that gene conversion plays a major role in APRT gene reversion in HTD114 cells. Therefore, the data demonstrate that BPDE can function as an effective frameshift mutagen in human cells.

Repair of benzo(a)pyrene diol epoxide- and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells.

Using Uvr proteins we have quantified benzo(a)pyrene diol epoxide (BPDE)-DNA adduct formation and repair at the dihydrofolate reductase (DHFR) and adenine phosphoribosyltransferase (APRT) genes in two Chinese hamster ovary cell lines: B-11 cells, which are 50-fold amplified for DHFR, and AT3-2 cells, which are diploid for DHFR. We have found that: 1) BPDE-DNA adduct formation in different regions of the DHFR gene is proportional to the concentration of BPDE. 2) There is no significant difference in the repair of BPDE-DNA adducts between the coding and noncoding regions in either amplified or nonamplified DHFR gene domains. 3) Repair in the nonamplified DHFR gene is more efficient (30-40%) than in the amplified DHFR genes. 4) There are no significant differences of repair in the transcribed or nontranscribed strands of the DHFR gene. 5) BPDE-DNA adduct formation and repair in the APRT gene in B-11 and AT3-2 cells are the same. These results contrast those for the repair of cyclobutane pyrimidine dimers, which occurs preferentially in the transcribed strand of the DHFR gene and in which gene amplification appears to play no role.

Nucleosome structure modulates benzo[a]pyrenediol epoxide adduct formation.

We have studied the binding of a chemical carcinogen to DNA reconstituted with histone octamers to determine the effect that nucleosome structure has on covalent adduct formation. Reconstitution of a plasmid containing the somatic 5S rRNA gene from Xenopus borealis resulted in characteristic nucleosome structure, as determined by micrococcal nuclease digestion, shifted migration in agarose gels, and hydroxyl radical footprinting. Formation of covalent adducts by benzo[a]pyrenediol epoxide (BPDE) occurred initially at a slower rate in reconstituted DNA than in naked plasmid, but after 2 h the total adduction levels (adducts/plasmid) were equal in both samples. Analysis of adduction at the sequence level by primer extension indicated that, after a 2-h BPDE reaction, the degree of adduction within the 5S rRNA nucleosome was suppressed by approximately 50% compared to naked DNA. The rotational setting of the guanines on the helix did not explain the level of adduction observed, since guanines in close proximity to the histone core were equally susceptible to adduction as guanines on the outer nucleosome surface. At early reaction times with BPDE, however, sequences near the 5S nucleosome dyad, where known modulations in the minor groove width occur, were the least susceptible to adduction. These results indicate that the structural features of DNA assembled into nucleosomes contribute to the susceptibility of the DNA to modification by BPDE.

Interactions between DNA molecules bound to RecA filament. Effects of base complementarity.

To gain information about the mechanism of RecA-promoted strand exchange reactions in genetic recombination, we have investigated the environment of and interactions between DNA strands accommodated in complexes with RecA protein. DNA bound in different sites in the RecA filament was tested by adding stoichiometric amounts of DNAs of variable base compositions. For this purpose, poly(dA) labeled by fluorescent benzo(a)pyrenediol epoxide (BPDE), which binds covalently to N-6 of adenine, was used. The fluorescence intensity, anisotropy, and quenching by acrylamide provide information about the DNA environment in the complexes with RecA. In the absence of extra DNA, binding of RecA to BPDE-poly(dA) only slightly affects both the intensity of the BPDE fluorescence and the accessibility of BPDE to acrylamide. However, a strongly increased fluorescence anisotropy shows that the mobility of the BPDE fluorophore is restricted in the RecA complex. Binding of a second and third single-stranded DNA to the RecA.BPDE-poly(dA) complex reduces the fluorescence intensity in a manner that depends on base complementarity. The change is large when the second and third DNAs are poly(dT), i.e. complementary to the already bound poly(dA) strand. In such a complex, the accessibility of BPDE to quencher is also reduced. When BPDE-poly(dA) was added as the second or third DNA in the RecA filament, the fluorescence intensity became smaller when the earlier bound DNA was complementary to the added DNA. These results indicate that all three DNA strands are interacting with each other in the RecA filament. BPDE-modified poly(dA) forms a duplex with poly(dT), whereupon the fluorescence of BPDE is strongly decreased. Binding of RecA to this duplex DNA increases the BPDE fluorescence, suggesting a destabilization of the duplex. Finally, we also find indications of base complementary interactions between single-stranded and double-stranded DNAs bound to the RecA filament.

Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts and increased p53 protein in mouse skin.

p53 protein expression has been shown to increase in response to DNA damage in cell culture systems. We have studied p53 expression and benzo[a]pyrene (B[a]P)-induced DNA-damage in the form of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts as measured by synchronous fluorescence spectrophotometry (SFS) in B[a]P-treated C57BL/6 mouse skin. Polyclonal murine antibody CM5, which is comparable to human CM1, detecting both wild-type and mutated protein, was used. BPDE-DNA adducts reached their maximum at 24 h after all dosage regimens, but were very well detectable also at 12 and 48 h after the treatment, while no adducts were measurable at 1 week and thereafter. p53 expression was seen in 9/17 (53%) skin samples from mouse treated with 500 microgram of B[a]P 12-48 h after the treatment, while all 25 (100%) cases of similarly treated mouse skins were negative after 30 weeks of the treatment. Only one positive sample of total 11 was found among mice treated with repeated 62.5 micrograms doses and this was 24 h after the last treatment. After one 62.5 micrograms dose all mice were negative. This is the first report of an association of p53 protein with DNA damage in vivo and gives support for the putative function of p53 in cellular defense machinery towards chemical damage.

Separation and characterization of post-labeled DNA adducts of stereoisomers of benzo[a]pyrene-7,8-diol-9,10-epoxide by immobilized boronate chromatography and HPLC analysis.

The carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) is enzymatically activated in cells to an ultimate carcinogenic metabolite, benzo[a]pyrene-7,8-dihydrodiol-9,10- epoxide (BaPDE), which reacts with DNA to form covalent adducts involved in the initiation of cancer. Previously, a post-labeling procedure that uses adenosine-5'-O-(3'-[35S]-thiotriphosphate) was developed to facilitate adduct analysis by HPLC. The much greater carcinogenic potency of (+)-anti-BaPDE makes it essential to be able to separate and identify the adducts formed by all four BaPDE enantiomers in DNA of cells exposed to BaP. Reversed-phase HPLC (RPHPLC) resolved the major (+)-anti-BaPDE-N2-deoxyguanosine [(+)-anti-BaPDE-N2-dG] adduct from the (+)-syn-BaPDE-N2-dG adduct. However, anti-BaPDE-N2-dG adducts formed by (+)- and (-)-anti-BaPDE were not resolved. By using ion-pair RPHPLC (IP-RPHPLC) with tetrabutylammonium phosphate, the [35S]post-labeled (-)-anti-BaPDE-N2-dG adduct eluted 3 min prior to the [35S]labeled (+)-anti-BaPDE-N2-dG adduct. In contrast, the major syn-BaPDE-N2-dG adducts were resolved better by RPHPLC than by IP-RPHPLC. The difference in conditions required for optimal separation of anti- and syn-BaPDE-DNA adducts necessitated the development of an immobilized boronate chromatography technique for the separation of anti- from syn-BaPDE-DNA adducts prior to analytical HPLC analysis. At 4 degrees C and with elution buffers containing high salt concentrations, the [35S]post-labeled anti-BaPDE-DNA adducts were selectively retained by a boronate column whereas the [35S]labeled syn-BaPDE-DNA adducts were not. Analysis of the multiple BaP-DNA adducts formed in BaP-treated hamster embryo cells by these techniques gave results comparable to those obtained by other methods. The major BaP-DNA adducts were anti-BaPDE-N2-dG, 14% from (-)- and 86% from (+)-anti-BaPDE. The ability of these techniques to detect low levels of PAH-DNA adducts because of the high specific radioactivity of 35S and to separate the DNA adducts formed by stereoisomeric PAH diol epoxides adducts by boronate by stereoisomeric PAH diol epoxides adducts by boronate chromatography and HPLC will facilitate studies of the role of individual PAH-DNA adducts in the induction of biological effects such as toxicity and carcinogenesis.

Involvement of P450 1A1 in Benzo(a)Pyrene but Not in Benzo(a)Pyrene‐7,8‐Dihydrodiol Activation by 3‐Methylcholanthrene‐Induced Mouse Liver Microsomes

Synchronous fluorescence spectrophotometry for benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts was used to study the activation pathway of benzo(a)pyrene in C57BL/6 mice. Benzo(a)pyrene but not benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced mouse liver microsomes was inhibited by a monoclonal antibody (Mab 1-7-1) against CYP1A1/2 suggesting that 1A1 probably takes part in the first P450 reaction. However, aryl hydrocarbon hydroxylase activity, a classical measure of benzo(a)pyrene metabolism, was not inhibited by the same concentration of Mab 1-7-1. None of the other antibodies used, detecting 2A, 2B, 2C or 2E subfamilies, inhibited the adduct formation. Troleandomycin and gestodene, chemical inhibitors of human 3A4, inhibited benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced microsomes to some extent only in high concentrations. Although liver microsomes from 3-methylcholanthrene-induced mice catalyzed the formation of BPDE-DNA in vitro clearly more than uninduced microsomes, 3-methylcholanthrene pretreatment in vivo decreased the adduct formation in benzo(a)pyrene-treated mice. These results emphasize the significance of detoxicating and DNA-repairing pathways in vivo. Finally, synchronous fluorescence spectrophotometry for BPDE-DNA measures the end-point of the three-step activation pathway while aryl hydrocarbon hydroxylase measures a one-step hydroxylation. Thus, these methods should be used rather as corroborative than mutually exclusive assays.

Immunoaffinity concentration of human lung DNA adducts using an anti-benzo[ a]pyrene-diol-epoxide-DNA antibody. Analysis by 32P-postlabelling or ELISA

DNA, isolated from 15 human lung autopsy samples, was examined for the presence of polycyclic aromatic hydrocarbon (PAH) DNA adducts. Using the nuclease P1 modification of the 32P-postlabelling technique, between 1 and 12 adducts/10 8 nucleotides were detected prior to immunoconcentration. Autoradiograms from most of the samples revealed a diagonal smear of radioactivity consistent with complex mixture (cigarette smoking) DNA damage. The DNA samples were digested to oligonucleotides, made single-stranded and subsequently applied to immunoaffinity columns containing immobilised anti-benzo[ a]pyrene (B(a)P)-7,8-diol-9,10-epoxide (BPDE) DNA polyclonal rabbit antibody. The material remaining bound to the column, in addition to that passing through, was analysed using both ELISA and 32P-postlabelling techniques. Column-bound adducts comprised between 0% and 78% of any particular sample. Immunoconcentration, followed by 32P-postlabelling of the material which had been bound to the column, revealed the presence of a number of discrete adduct spots in autoradiograms of the more heavily adducted samples. Sample DNA not retained by the columns was also analysed; the chromatographic pattern obtained was a dense zone of radioactive material migrating from the origin. This evidence suggests that the composition of PAH-DNA adducts found in human lung samples exhibits wide inter-individual variation.

Pulmonary alveolar macrophages in molecular epidemiology and chemoprevention of cancer.

In addition to possessing an extraordinary sweeping activity, pulmonary alveolar macrophages (PAM) are equipped with the biochemical mechanisms involved in the metabolism of carcinogens, which were found to be inducible in humans by cigarette smoke. Moreover, several defense processes were stimulated in rat PAM after in vivo administration of the anticarcinogen N-acetylcysteine (NAC). Benzo[a]pyrene diol epoxide (BPDE)-DNA adducts, as revealed by synchronous fluorescence spectrophotometry, were selectively detected in PAM of smokers and persisted up to 6 months. The amount of adducts was significantly correlated with the number of currently smoked cigarettes but not with the cigarettes smoked in a lifetime (pack-years). Nevertheless, deviations from the regression line pointed out the role of interindividual variability factors in adduct formation. Probably due to the low mitotic rate of PAM in the respiratory lumen, the frequency of micronuclei was not enhanced in smokers. However, parallel assays in rats showed that micronuclei can be enhanced after massive intratracheal administration of benzo[a]pyrene or whole-body exposure to high amounts of mainstream cigarette smoke, which also induced BPDE-DNA adducts in lung cells and other organs, including the heart. All these adverse effects were markedly inhibited by the oral administration of NAC, which provides the premise and rationale for a future study on the protective effects of oral NAC in heavy smokers.