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PubMed1996

Disposition and biological activity of benzo[a]pyrene-7,8-dione. A genotoxic metabolite generated by dihydrodiol dehydrogenase.

A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-dihydrodiols to yield o-quinones, a reaction catalyzed by dihydrodiol dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo-[a] pyrene (BP-diol) was oxidized by this route to yield benzo [a] pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [3H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 +/- 17 adducts/ 10(6) base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes treated with either BP-diol or BPQ were found to produce large quantities of superoxide anion radical (O2.-). The amount of O2.- generated by BP-diol was blocked by DD inhibitors. These data suggest that by diverting BP-diol to BPQ reactive oxygen species (ROS) were generated which caused DNA fragmentation. The ability of BPQ to cause DNA strand scission was further studied using supercoiled phi X174 DNA. It was found that BPQ caused concentration-dependent (0.05-10 microM) strand scission in the presence of 1 mM NADPH (which promoted redox-cycling) provided CuCl2 (10 microM) was present. Complete destruction of the DNA was observed using 10 microM BPQ. This strand scission was prevented by catalase and hydroxyl radical scavengers but not by superoxide dismutase. These data indicate that ROS were responsible for the destruction of the DNA. Using 20 microM (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene [(+/-)-anti-BPDE] only single nicks in the DNA were observed indicating that BPQ was the more potent chemical nuclease. BPQ was also found to be a direct-acting mutagen in the Ames test using Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA104, but was 10-5500-fold less efficient as a mutagen than (+/-)-anti-BPDE. Our data indicate that DD suppresses the mutagenicity of (+/-)-anti-BPDE by producing BPQ, but in doing so a potent chemical nuclease is produced which causes extensive DNA fragmentation via the generation of ROS.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/dA adduct: glycosidic rotation from syn to anti at the modified deoxyadenosine.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

Interactions between DNA polymerase beta and the major covalent adduct of the carcinogen (+)-anti-benzo[a]pyrene diol epoxide with DNA at a primer-template junction.

A molecular dynamics simulation has been carried out with DNA polymerase beta (beta pol) complexed with a DNA primer-template. The templating guanine at the polymerase active site was covalently modified by the carcinogenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, to form the major (+)-trans-anti-benzo[a]pyrene diol epoxide covalent adduct. Thus, the benzo[a]pyrenyl moiety (BP) is situated in the single-stranded template at the junction between double- and single-stranded DNA. The starting structure was based on the X-ray crystal structure of the rat beta pol primer-template and ddCTP complex [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903]. During the simulation, the BP and its attached templating guanine rearrange to form a structure in which the BP is closer to parallel with the adjacent base pair. In addition, the templating attached guanine is displaced toward the major groove side and access to its Watson-Crick edge is partly obstructed. This structure is stabilized, in part, by new hydrogen bonds between the BP and beta pol Asn279 and Arg283. These residues are within hydrogen bonding distance to the incoming ddCTP and templating guanine, respectively, in the crystal structure of the beta pol ternary complex. Site-directed mutagenesis has confirmed their role in dNTP binding, discrimination, and catalytic efficiency [Beard, W. A., Osheroff, W. P., Prasad, R., Sawaya, M. R., Jaju, M., Wood, T. G., Kraut, J., Kunkel, T. A., and Wilson, S. H. (1996) J. Biol. Chem. 271, 12141-12144]. The predominant biological effect of the BP is DNA polymerase blockage. Consistent with this biological effect, the computed structure suggests the possibility that the BP's main deleterious impact on DNA synthesis might result at least in part from its specific interactions with key polymerase side chains. Moreover, relatively modest movement of BP and its attached guanine, with some concomitant enzyme motion, is necessary to relieve the obstruction and permit the observed rare incorporation of a dATP opposite the guanine lesion.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1999

RNA tertiary folding monitored by fluorescence of covalently attached pyrene.

The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4-P6 domain was used as a test system. Guided by the P4-P6 X-ray crystal structure, we chose a nucleotide (U107) for which derivatization at the 2'-position should not perturb the folded conformation. A 15-mer RNA oligonucleotide with a 2'-amino substitution at U107 was derivatized with a pyrene chromophore on a variable-length tether, and then ligated to the remainder of P4-P6, providing a site-specifically pyrene-labeled P4-P6 derivative. Upon titration of the pyrene-derivatized P4-P6 with Mg(2+), the equilibrium fluorescence intensity reversibly increased several-fold, as expected if the probe's chemical microenvironment changes as the RNA to which it is attached folds. The concentration and specificity of divalent ions required to induce the fluorescence change (Mg(2+) approximately Ca(2+) > Sr(2+)) correlated well with biochemical folding assays that involve nondenaturing gel electrophoresis. Furthermore, mutations in P4-P6 remote from the chromophore that shifted the Mg(2+) folding requirement on nondenaturing gels also affected in a predictable way the Mg(2+) requirement for the fluorescence increase. Initial stopped-flow studies with millisecond time resolution suggest that this fluorescence method will be useful for following the kinetics of P4-P6 tertiary folding. We conclude that a single site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equilibrium progress and the real-time kinetics of RNA tertiary folding.

Animals↗
PubMed2002

Sorption of pyrene to dissolved humic substances and related model polymers. 2. Solid-phase microextraction (SPME) and fluorescence quenching technique (FHT) as analytical methods.

Sorption coefficients for pyrene on dissolved humic substances and on poly(acrylic acid) esters as well-defined model polymers were determined using solid-phase microextraction (SPME) and the fluorescence quenching technique (FQT). The results of both analytical methods were compared and theoretically evaluated, which led to the conclusion that the sorption coefficients measured by SPME and FQT are inevitably different: SPME measures activity-based and FQT concentration-based sorption coefficients. The environmental relevance of the two types of sorption coefficients is discussed. FQT is inappropriate to measure sorption coefficients for pyrene with the synthetic sorbents. Inspection of the vibrational structure of the fluorescence spectra of those solutions indicates a highly hydrophobic microenvironment of pyrene. This can be explained by an intra- or intermolecular agglomeration of hydrophobic moieties forming a favorable host for hydrophobic solutes.

Adsorption↗
PubMed2004

Investigation of sorption behavior between pyrene and colloidal organic carbon from activated sludge processes.

The sorption behavior of pyrene for different size fractions of colloidal organic carbon (COC) originating from two biological wastewater treatment facilities (a full-scale activated sludge system (FSAS) and a membrane bioreactor (MBR)) was investigated by fluorescence quenching. Fluorescence lifetime measurements demonstrated a dynamic quenching component in all samples, including the colloidal-free filtrates. COC sorption coefficients (Kcoc) for pyrene ranged from <1 x 10(3) L/kgcoc to 80 x 10(3) L/kgcoc and were comparable to values obtained in the literature for natural organic matter. Both linear and nonlinear behaviors were observed in the Freundlich-described converted isotherms (n = 0.89-2.1). The aromatic content of COC was quantified by the molar extinction coefficient at 280 nm (e280). Good correlations were observed between COC-pyrene sorption coefficients and e280 coefficients, suggesting that e280 may be a useful tool for predicting colloidal transport of hydrophobic organic compounds (HOCs) from activated sludge systems. The removal of COC from treated effluents may appreciably reduce the concentrations of HOCs discharged to receiving streams.

Adsorption↗
PubMed2004

Influence of humic substance adsorptive fractionation on pyrene partitioning to dissolved and mineral-associated humic substances.

Changes in pyrene binding by dissolved and mineral-associated humic substances (HS) due to HS adsorptive fractionation processes were examined in model environmental systems using purified Aldrich humic acid (PAHA) and Suwannee River fulvic acid (SRFA). For PAHA, carbon-normalized pyrene binding coefficients for nonadsorbed, residual fractions (Koc(res)) were different from the original dissolved PAHA Koc value (Koc(orig)) prior to contact with the mineral suspensions. A strong positive correlation between pyrene log Koc(res) and log weight-average molecular weight (MWw) for residual PAHA fractions was observed, which was relatively independent of the specific mineral adsorbent used and hypothesized fractionation processes. A strong positive correlation between log Koc(ads) and log MWw was also found for PAHA fractions adsorbed to kaolinite at low mass fraction organic carbon levels, although the relationship was statistically different from the one found with residual PAHA fractions. The same trends and correlations found for PAHA were not observed with SRFA, suggesting that the impacts of HS adsorptive fractionation on changes in hydrophobic organic contaminants binding are also influenced by the source and other biogeochemical characteristics of HS.

Adsorption↗
PubMed2005

Phenanthrene and pyrene sorption and intraparticle diffusion in polyoxymethylene, coke, and activated carbon.

We report sorption isotherms and uptake kinetics for phenanthrene and pyrene with three organic model sorbents: polyoxymethylene (POM), coke, and activated carbon. We combine batch equilibration and kinetic experiments with the direct observation of the long-term diffusion of phenanthrene and pyrene as measured within cross-sectioned particles using microprobe laser-desorption laser-ionization mass spectroscopy (muL2MS). For POM pellets, the intraparticle concentration profiles predicted from kinetic batch experiments and a polymer diffusion model with spherical geometry are in agreement with the independent muL2MS measurements. For coke particles, the apparent diffusivities decreased with smaller particle size. These trends in diffusivities were described by a sorption-retarded pore diffusion model with a particle-size-dependent solid-water partitioning coefficient obtained from apparent equilibrium observed in the kinetic batch studies. For activated carbon, the muL2MS measurements showed faster radial diffusion of phenanthrene and pyrene into the particle interior than predicted from diffusion models based on a single sorption domain and diffusivity. A branched pore kinetic model, comprising polycyclic aromatic hydrocarbon (PAH) macropore diffusion with kinetic exchange of PAH between macroporous and microporous domains, fits the experimental observations better. Because of parallel macro- and microdiffusion processes, nonlinear sorption isotherms, and a concentration-dependent diffusivity, it is not possible to make independent parameter estimations for intraparticle diffusion in activated carbon using our present procedures.

Adsorption↗
PubMed2005

Nitration of benzo[a]pyrene adsorbed on coal fly ash particles by nitrogen dioxide: role of thermal activation.

Nitration of benzo[a]pyrene (BaP) by nitrogen dioxide (NO2) adsorbed on the surface of thermally activated coal fly ash and model aluminosilicate particles led to the formation of nitrobenzo[a]pyrenes as verified by extraction and gas chromatography/mass spectrometry (GC/MS). In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) was utilized to follow the nitration reaction on the surface of zeolite Y. Nitrobenzo[a]pyrene formation was observed along with the formation of nitrous acid and nitrate species. The formation of the BaP radical cation was also observed on thermally activated aluminosilicate particles by electron spin resonance (ESR) spectroscopy. On the basis of GC/MS, DRIFTS, and ESR spectroscopy results, a mechanism of nitration involving intermediate BaP radical cations generated on thermally activated aluminosilicate particles is proposed. These observations have led to the hypothesis that nitration of adsorbed polyaromatic hydrocarbons on coal fly ash by reaction with nitrogen oxides can occur in the smokestack, but with the aging of the fly ash particles, the extent of the nitration reaction will be diminished.

Adsorption↗
PubMed2001

Stereochemical, structural, and thermodynamic origins of stability differences between stereoisomeric benzo[a]pyrene diol epoxide deoxyadenosine adducts in a DNA mutational hot spot sequence.

Benzo[a]pyrene (BP), a prototype polycyclic aromatic hydrocarbon (PAH), can be metabolically activated to the enantiomeric benzo[a]pyrene diol epoxides (BPDEs), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the (-)-(7S,8R,9R,10S) enantiomer. These can react with adenine residues in DNA, to produce the stereoisomeric 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts. High-resolution NMR solution studies indicate that in DNA duplexes the 10R (-) adduct is intercalated on the 5'-side of the modified adenine, while the 10S (+) adduct is disordered, exhibits multiple adduct conformations, and is positioned on the 3'-side of the modified adenine. Duplexes containing the 10S (+) adduct positioned at A within codon 61 of the human N-ras sequence CAA are thermodynamically less stable and more easily excised by human DNA repair enzymes than those containing the 10R (-) adduct. However, the molecular origins of these differences are not understood and represent a fascinating opportunity for elucidating structure-function relationships. We have carried out a computational investigation to uncover the structural and thermodynamic origins of these effects in the 11-mer duplex sequence d(CGGACAAGAAG).d(CTTCTTGTCCG) by performing a 2-ns molecular dynamics simulation using NMR solution structures as the basis for the starting models. Then, we applied the MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method to compute free energy differences between the stereoisomeric adducts. The 10R (-) isomer is more stable by approximately 13 kcal/mol, of which approximately 10 kcal/mol is enthalpic, which agrees quite well with their observed differences in thermodynamic stability. The lower stability of the 10S (+) adduct is due to diminished stacking by the BP moiety in the intercalation pocket, more helix unwinding, and a diminished quality of Watson-Crick base pairing. The latter stems from conformational heterogeneity involving a syn-anti equilibrium of the glycosidic bond in the modified adenine residue. The lower stability and conformational heterogeneity of the 10S (+) adduct may play a role in its enhanced susceptibility to nucleotide excision repair.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2004

Reduction of dioxygen catalyzed by pyrene-wired heme domain cytochrome P450 BM3 electrodes.

We have electronically wired the cytochrome P450 BM3 heme domain to a graphite electrode with the use of a pyrene-terminated tether. AFM images clearly reveal that pyrene-wired enzyme molecules are adsorbed to the electrode surface. The enzyme-electrode system undergoes rapid and reversible electron transfer, displaying a standard rate constant higher than that of any other P450-electrode system. We also show that the graphite-pyrene-BM3 system catalyzes the four-electron reduction of dioxygen to water.

Bacterial Proteins↗
PubMed2004

Thermal and photochemistry of a pyrene dihydrodioxin (PDHD) and its radical cation: a photoactivated masking group for ortho-quinones.

Pyrene dihydrodioxins (1 and 2) have been synthesized and shown to be effective photochemical blocking groups for pyrene-4,5-dione (3). The mechanism of quinone release proceeds through the formation of a remarkably stable radical cation. Direct evidence is provided that this radical cation is not only thermally labile but also photochemically labile, and that both pathways lead to quinone extrusion. Once initiated with UV light, the pyrene quinone product serves as an electron-transfer photosensitizer for the further release of quinone with visible light.

Cations↗
PubMed2006

Supramolecular probe for bicarbonate exhibiting anomalous pyrene fluorescence in aqueous media.

Highly selective HCO3- sensing is realized in a pH 8.6 aqueous solution on the basis of the HCO3--induced anomalous pyrene fluorescence of an association dimer that consists of pyrene-appended gamma-cyclodextrins having a triamine linker. The anomalous fluorescence that is produced by the HCO3--induced twisted conformation of the pyrene residues in the association dimer is insensitive to the presence of other anions.

Bicarbonates↗
PubMed2006

Structure and vibrational spectra of mononitrated benzo[a]pyrenes.

The molecules benzo[a]pyrene (BaP) and 1-, 3-, and 6-nitrobenzo[a]pyrene (1-NBaP, 3-NBaP, 6-NBaP) are currently of significant interest due to their presence in respirable combustion exhaust particulates and their mutagenic and carcinogenic properties. Structure-function correlations as well as spectroscopic signatures for trace analysis are necessary for these benzo[a]pyrene derivatives. In this paper, detailed infrared and Raman spectroscopic data of BaP and its three mononitrated isomers are provided for the first time. By utilizing density functional theory (DFT, B3LYP method with 6-311+G basis set), the molecular geometries and the vibrational spectra are calculated. Good agreement is noted between the calculated and experimental geometry for BaP, and predictions of the vibrational data for all compounds are within approximately 5 cm-1 of the experimental data. Normal mode assignments are proposed with particular emphasis on the nitro group vibrations. The geometrical distortions of the BaP structure upon nitro group substitution and correlations between structural parameters and vibrational data as well as structure-function relationships related to the mutagenicity of this important class of polycyclic aromatic hydrocarbons are discussed.

Air Pollutants↗
PubMed2001

Solvent-free synthesis of benzo[a]pyrene 7,8-diol 9,10-epoxide adducts at the N(2)-position of deoxyguanosine.

[reaction: see text] The first solid-state (or solvent-free) synthesis of protected deoxyguanosine (dG) adducts of benzo[a]pyrene diol epoxides at room temperature is reported. Whereas dG adducts derived from cis- and trans-opening of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-1 1) are formed as a 1:1 mixture, the direct opening of the diastereomeric (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-2, 2) produced a 15:85 ratio favoring the trans-opened dG adduct 7.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Detection and quantification of depurinated benzo[a]pyrene-adducted DNA bases in the urine of cigarette smokers and women exposed to household coal smoke.

Polycyclic aromatic hydrocarbons (PAH) are metabolized to electrophiles that can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAH-treated mouse skin. Depurinating adducts account for 71% of all DNA adducts formed in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls for the presence and quantities of the depurinated BP-adducted DNA bases, 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade). Since these adducted bases originate from reaction of the BP radical cation with double-stranded DNA and not with RNA or denatured DNA, their presence in urine is indicative of DNA damage. Urine samples were fractionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis with laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to coal smoke, but were not detected in urine from the 13 control subjects. Concentrations were estimated to be 60-340 and 0.1-0.6 fmol/mg of creatinine equivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20-300 times the concentration of BP-6-N7Ade in the urine of coal smoke-exposed women, but was not detected in the urine of cigarette smokers. This difference may be due to the remarkably different BP exposures experienced by the two groups of PAH-exposed individuals. These results justify more extensive studies of depurinated BP-adducted DNA bases as potential biomarkers of PAH-associated cancer risk.

Adult↗
PubMedDate not supplied

Fluorescence line narrowing spectrometric analysis of benzo[a]pyrene-DNA adducts formed by one-electron oxidation.

Fluorescence line narrowing (FLN) was demonstrated for five benzo[a]pyrene (BP)-nucleoside adducts synthesized by one-electron oxidation of BP in the presence of guanosine, deoxyguanosine, and deoxyadenosine. The standard FLN spectra were used to prove that a major depurination adduct from the binding of BP to DNA in rat liver nuclei is 7-(benzo[a]pyren-6-yl)guanine (N7Gua). The structural characterization was performed with only 20 pg of the adduct. Metabolic activation of BP by one-electron oxidation in the horseradish peroxidase catalyzed reaction of BP with DNA (in vitro) was also investigated. The major adduct identified was 8-(benzo[a]pyren-6-yl)guanine (C8Gua).

Animals↗
PubMedDate not supplied

Immunologic methods for the detection of benzo[a]pyrene metabolites in urine.

Two monoclonal antibodies (10C10 and 4D5) have been developed from the spleen cells of Balb/c mice immunized with 6-aminobenzo[a]pyrene covalently coupled to bovine serum albumin. These antibodies have been used in an immunoassay for the detection of benzo[a]pyrene and its metabolites in mouse urine. The antibodies were characterized in terms of sensitivity and specificity by competitive enzyme-linked immunosorbent assay (ELISA). With both antibodies, 50% inhibition of antibody binding is at 4 pmol of BP. The antibodies also cross-react with a number of BP metabolites as well as with several other polycyclic aromatic hydrocarbons (PAHs) including pyrene, 1-aminopyrene, and 7,12-dimethylbenz[a]anthracene but with different sensitivities. These results suggest that this assay will detect multiple PAH metabolites in urine. To test the assay on biological samples, mice were treated with [3H]BP, and urine was collected and digested with beta-glucuronidase and aryl sulfatase. Several methods were used to isolate BP and its metabolites from the urine, including ethyl acetate extraction, Sep-pak C18 cartridge chromatography, XAD2 resin chromatography, and immunoaffinity chromatography with antibody 4D5. Analysis of the urine extracts with antibody 4D5 gave 50% inhibition at 12-15 pmol of metabolites. Thus, quantitation of metabolites in this sample by competitive ELISA against a standard curve of BP would have underestimated actual metabolite levels by about 70%. This assay will be applied to the analysis of urines from individuals with environmental or occupational exposure. Since humans are usually exposed to BP in complex mixtures of PAHs, multiple metabolites may be present in the urine, making absolute quantitation difficult. This assay should thus serve as a general indicator of exposure to this class of chemicals.

Animals↗