Highly diastereoselective synthesis of nucleoside adducts from the carcinogenic benzo[a]pyrene diol epoxide and a computational analysis

A diastereoselective synthesis of the nucleoside adducts corresponding to a cis ring-opening of the carcinogen (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE-2) by 2'-deoxyadenosine and 2'-deoxyguanosine is described. The key intermediate (+/-)-10alpha-amino-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene was synthesized by a highly diastereoselective dihydroxylation wherein phenylboronic acid was a water surrogate. The resulting boronate ester was converted to a tetraol derivative in which two of the four hydroxyl groups (trans 7, 8) were protected as benzoate esters while the remaining two (cis 9, 10) were free. The cis glycol entity was then subjected to a reaction with 1-chlorocarbonyl-1-methylethylacetate to yield an intermediate chloro monoacetoxy dibenzoate. Displacement of the halide with azide, complete cleavage of the esters, and catalytic reduction of the azide yielded the requisite amino triol. Fluoride displacement from appropriately protected nucleoside derivatives, 6-fluoropurine 2'-deoxyribonucleoside and 2-fluoro-2'-deoxyinosine, by the amino triol then yielded diastereomeric pairs of diol epoxide-adducted 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) nucleosides. Small aliquots of these adducts were separated for characterization purposes. The present approach provides the first diastereoselective synthesis of the cis adducts of BaP DE-2 with 2'-deoxyguanosine as well as the first synthesis of both dA and dG adducts from a common intermediate. An informative analysis of the 1H NMR spectra of the cis adducts synthesized and comparisons to the trans adducts are reported. To gain insight into the diastereoselectivity in the key dihydroxylation step, a computational analysis, including molecular mechanics (MMFF94) and semiempirical AM1 geometry optimizations, yielded results that are in fairly good agreement with the experimental observations.     

A rapid method for isolation of human hemoglobin benzo[a]pyrene diol epoxide derived adducts using high performance liquid chromatography

A method is described to isolate rapidly human hemoglobin-benzo[a]pyrene diol epoxide adducts. A combination of 300 A pore size C4 reversed phase HPLC to effect separation of adducted protein from native protein, and mu-bore C18 reversed phase HPLC to isolate and partially characterize proteolytic peptide adducts (by UV), was used.     

Wavefunction and reactivity study of benzo[a]pyrene diol epoxide and its enantiomeric forms

Benzo[a]pyrene is a known carcinogen, which derives from fossil fuel combustion, cigarette smoke, and generic biomass combustion including traffic emissions. This potent carcinogen has a well-known mechanism of action, leading to the formation of adducts with the DNA, primarily at guanosine positions. The reactivity and chemistry of this notorious compound are, however, dependent on the electronic configuration of the biologically activated metabolite, the benzo[a]pyrene diol epoxide. The activated metabolite exists mainly as four isomers, which have particular chemical reactivities toward guanosine sites on the DNA. These isomers exert also a different carcinogenicity compared to one another, which is a feature that is conventionally attributed to their geometry. However, the reactivity and properties of the isomers are not fully defined, and a determination of these properties by wavefunction behavior is required. This study reports the electronic properties of the benzo[a]pyrene diol epoxide enantiomers, along with a detailed analysis of the energy landscape, geometry, and electronic configuration of the epoxide ring. The results show that the epoxide ring, the core of the reactivity, bears different properties at the level of wavefunction for each isomer. Each of the isomers has a distinct profile on the epoxide ring, in terms of hydrogen bonds and in terms of the non-covalent interaction between the diol groups and the epoxide. These profiles generate differential reactivities of epoxide group, which can be attributed to its local bond lengths, the electron localization function, and polarized bonds. Most interestingly, the quantum chemical calculations showed also that the epoxide ring is inclined more perpendicularly toward the angular ring plane for the more carcinogenic isomers, a feature which suggests a potential geometrical relationship between the inclination of the epoxide group and its interaction with the guanosine group upon adduct formation. Our results introduce novel and crucial information, which assist in understanding the mechanism of toxic potential of this known molecule, and display the strength and level of detail of applying quantum chemical methods to reveal the reactivity, energy properties, and electronic properties of a mutagen.     

Reaction of cyclohexene oxides with phosphodiesters - towards understanding the reaction of benzo[a]pyrene diol epoxide with DNA

Phosphodiesters react with cyclohexene oxides to give phosphotriesters with high stereo- and regiospecificity.     

On-line identification of diastereomeric dibenzo[a,l]pyrene diol epoxide-derived deoxyadenosine adducts by capillary electrophoresis-fluorescence line-narrowing and non-line narrowing spectroscopy.

A capillary electrophoretic method for the separation and on-line identification of closely related analytes using low-temperature fluorescence spectroscopy is reported for the eight diastereomeric deoxyadenosine (dA) adducts derived from dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE). Electrophoretic separation of stereoisomers was accomplished by application of a mixed surfactant buffer [dioctyl sulfosuccinate (DOSS) and Brij-S], which was below the critical micelle concentration (CMC) due to the high concentration (approximately 25%) of organic solvent. Addition of multiple surfactant additives to the separation buffer provided electrophoretic resolution, which was unattainable under single surfactant conditions. It is shown that the CE-separated analyte zones could be identified on-line via low-temperature (4.2 K) fluorescence non-line narrowing and fluorescence line-narrowing (FLN) spectroscopy. In addition, it was determined that in CE buffer trans-syn-,cis-syn- and cis-anti-DB[a,l]PDE-14-N6dA diastereomeric adducts exist mostly with the -dA and DB[a,l]P moiety in an "open"-type conformation while the trans-anti-DB[a,l]PDE-14-N6dA adducts exist in two different conformations whose relative distribution depends on matrix composition. The above conformations have also been revealed by selective laser excitation. Thus, the low-temperature methodology not only provides fingerprint structure via vibrationally resolved 4.2 K fluorescence spectra for adduct identification, but also provides conformational information on the spatial relationship of the carcinogen and dA moiety. These results, taken together with those for DB[a,l]P-DNA adducts formed in standard glasses and mouse epidermis exposed to DB[a,l]P, support our earlier findings that DB[a,l]P-derived adducts exist in different conformations [Jankowiak et al., Chem. Res. Toxicol. 11 (1998) 674]. Therefore, the combination of the separation power of CE and spectral selectivity of low-temperature fluorescence spectroscopy at NLN and FLN conditions provides a powerful methodology which should prove useful for identification of closely related DNA adducts formed at low levels in biological systems.     

Quantification of benzo[a]pyrene diol epoxide DNA-adducts by stable isotope dilution liquid chromatography/tandem mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants found in car exhausts, charbroiled food, and tobacco smoke. Three pathways for the metabolic activation of B[a]P to ultimate carcinogens have been proposed. The most widely accepted pathway involves cytochrome-P450 (CYP) 1A1- and/or 1B1-mediated formation of B[a]P-7,8-oxide, which undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further CYP1A1- and/or CYP1B1-mediated activation of the dihydrodiol results in the formation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. In previous studies, it was demonstrated that (+)-anti-B[a]PDE was the most potent tumorigen of the CYP-derived B[a]PDE diastereomers. We have developed a stable isotope dilution, liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay for all eight (+/-)-anti-B[a]PDE-derived dGuo and dAdo DNA-adducts. The LC-MRM/MS assay was rigorously validated and used to show that (+)-anti-trans-B[a]PDE-dGuo was the major adduct formed when naked DNA and human bronchoalveolar adenocarcinoma H358 cells were treated with (+/-)-anti-B[a]PDE. The preference for DNA-adducts derived from (+)-anti-B[a]PDE was even more apparent in cellular DNA. Thus, the increased potency of (+)-anti-B[a]PDE as a tumorigen is most likely due its ability to preferentially form DNA-adducts when compared with (-)-anti-B[a]PDE. Also, the adduct profile suggests that this occurs by binding of (+)-anti-B[a]PDE to DNA in a manner that facilitates covalent binding to dGuo rather than dAdo residues.     

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.     

Analysis of benzo[a]pyrene diol epoxide-DNA adducts by capillary zone electrophoresis- electrospray ionization-mass spectrometry in conjunction with sample stacking

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.     

Selective digestion and novel cleanup techniques for detection of benzo[a]pyrene diol epoxide-DNA adducts by capillary electrophoresis/mass spectrometry

Benzo[a]pyrene (BP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) which, upon metabolic conversion to reactive benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), has been found to attach covalently to DNA. Given the low level of DNA adducts typically present in vivo or in vitro, an essential first step prior to capillary electrophoresis/mass spectrometry (CE/MS) (or liquid chromatography/mass spectrometry (LC/MS)) analysis of the DNA digests is the removal of the bulk non-adducted nucleotides, enzymes or salts, and isolation of enriched adducts. This report focuses on the development of novel sample handling methods aimed at facilitating the analysis of BPDE-DNA adducts by CE/MS. This approach involves a simple variation on the digestion procedure, in combination with the use of metal affinity ZipTips for the more efficient cleanup of BPDE-DNA adducts formed in vitro for subsequent CE/MS analysis. The previously described digestion procedure, consisting of micrococcal nuclease, spleen phosphodiesterase and nuclease P1, allows for selective dephosphorylation of normal nucleotides, while leaving adducted nucleotides intact. Metal affinity ZipTips, typically used for selective extraction of phosphopeptides, were used here for extraction of adducted nucleotides. The utility of metal affinity SPE was tested on mixtures of dG and dGp, wherein nucleotide extracts contained no detectable nucleosides by CE/UV analysis. An in vitro BPDE-DNA incubation was then digested using the above procedure. Metal affinity solid-phase extraction (SPE) was subsequently used for the selective isolation of phosphorylated components, i.e., adducted nucleotides, from the mixture of enzymes and non-adducted nucleosides. SPE extracts were enriched in nucleotide adducts and analyzed using sample stacking and CE/MS. This method has several advantages over previously described cleanup procedures for dGp-BPDE adducts: fast, simple, uses commercially available materials, no need for excessive dilution (small scale), the suitability for use with automation, and possible applicability to other bulky hydrophobic adducts.     

Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence. Analysis of A549 cells

Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.     

Preparation, identification and analysis of stereoisomeric anti-benzo[a]pyrene diol epoxide-deoxyguanosine adducts using phenyl liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection

Benzo[a]pyrene, a common environmental pollutant, can be metabolized into reactive anti-benzo[a]pyrene diol epoxide (anti-BPDE), which predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. To characterize the stereochemistry of these adduct isomers, preparation of single adducted deoxyguanosine (dG) is required for efficient enantiomeric analysis. Here, we demonstrate an improved method for preparation, identification, and analysis of four BPDE-adducted dGs, including (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-BPDE-N(2)-dG. These stereoisomerically adducted nucleosides were first synthesized by a direct reaction of (+/-)-anti-BPDE with dG, followed by optimized solid-phase extraction (SPE) and HPLC purification. The reaction of (+/-)-anti-BPDE and dG displayed a yield as high as 45%. The developed preparation method does not require any enzymatic digestion. Based on highly efficient separation achieved by optimization of stationary phase and mobile phase, LC-UV-MS/MS and LC-diode array detection (DAD)-fluorescence detection (FL) methods were established for characterization and analysis of the four stereoisomeric anti-BPDE-dGs. The established LC-DAD-FL method may provide characterization and analysis of four stereoisomeric anti-BPDE-dGs and two interfering anti-BPDE tetrols by taking advantage of their distinct fluorescence quenching.     

The solid-matrix phosphorescence of (±)-anti-dibenzo[a,l]pyrene diol epoxide-DNA adducts and benzo[e]pyrene

New solid-matrix phosphorescence (SMP) methods for (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P were developed. The methods can be used to detect and characterize (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P by employing SMP spectra, intensities, and lifetimes acquired with the heavy-atom salt, TlNO₃, on Whatman 1PS paper. With the SMP data, a number of photophysical parameters were calculated such as biexponential SMP decay curves, pre-exponential factors, and fractional contribution to SMP decay curves. The SMP results were compared with earlier SMP data for (±)-anti-BPDE-DNA adducts and tetrol I-1. The SMP results show that small molecular-weight compounds like B[e]P can be readily detected and characterized by SMP. For example, the limit of detection for B[e]P was 0.60 pmol. Comparison of the SMP properties of the (±)-anti-DB[a,l]PDE-DNA adducts with earlier SMP data for the (±)-anti-BPDE-DNA adducts showed major differences in the SMP spectra, intensities, and lifetimes. The methods developed are important for the comparison of the SMP properties of different diol epoxides of PAH bonded to DNA.     

Second-derivative constant-wavelength synchronous scan spectrofluorimetry for determination of benzo[b]fluoranthene, benzo[a]pyrene and indeno[1,2,3-cd]pyrene in drinking water

The use of derivative constant-wavelength synchronous scan fluorimetry is reported for the determination of three polycyclic aromatic hydrocarbon pollutants in drinking water (linearity range 0.4-4 mug 1(-1)). The limits of detection (LD) and quantification (LQ) (mug 1(-1)) are 0.01 and 0.07 for benzo[b]fluoranthene, 0.03 and 0.12 for benzo[a]pyrene and 0.19 and 0.57 for indeno[1,2,3-cd]pyrene in the presence of three other pollutants, benzo[k]fluoranthene, benzo[ghi]perylene and fluoranthene. The precision (RSD /= 85%) were satisfactory.     

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 (G₇) 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. Copyright © 2009 John Wiley & Sons, Ltd.     

The characterization of (+/-)-anti-benzo[a]pyrene diolepoxide-DNA adducts and (+/-)-anti-dibenzo[a,l]pyrene diolepoxide--DNA adducts in the same DNA sample using solid-matrix phosphorescence

Solid-matrix phosphorescence (SMP) spectra and lifetimes were used to characterize the (±)-anti-benzo[a]pyrene diolepoxide [(±)-anti-B[a]PDE] and (±)-anti-dibenzo[a,l]pyrene diolepoxide [(±)-anti-DB[a,l]PDE] bonded to the same sample of DNA. SMP spectra and lifetimes were also acquired for two samples of DNA that had only (±)-anti-B[a]PDE or (±)-anti-DB[a,l]PDE bonded to the individual samples of DNA. A detailed comparison of the SMP properties was made among the three samples of DNA. The SMP excitation spectra for the (±)-anti-B[a]PDE-DNA and the (±)-anti-DB[a,l]PDE-DNA adducts were very similar. However, the SMP emission spectra of the two DNA adduct systems were very dissimilar with a major emission band for the (±)-anti-B[a]PDE-DNA adducts appearing at 613 nm and for the (±)-anti-DB[a,l]PDE-DNA adducts a major band was at 558 nm. It was possible to selectively use SMP emission wavelengths and obtain a SMP excitation of spectrum of the (±)-anti-DB[a,l]PDE-DNA adducts in the dual adducted DNA sample without the (±)-anti-B[a]PDE-DNA adducts emitting SMP. In addition, it was shown that the SMP emission spectrum of the dual adducted DNA sample could be used to detect both adduct systems in the modified DNA sample. It was demonstrated that the SMP lifetimes could be effectively employed to characterize the dual adducted DNA sample. For example, the SMP decay curve for the (±)-anti-DB[a,l]PDE-DNA adducts could be acquired without any SMP emission from the (±)-anti-B[a]PDE-DNA adducts. Also, ln(SMP intensity) versus time plots were very useful in characterizing the dual adducted DNA sample.     

Simultaneous analysis of four stereoisomers of anti-benzo[a]pyrene diol epoxide-deoxyguanosine adducts in short oligodeoxynucleotides using reversed-phase high-performance liquid chromatography

anti-Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE), a reactive metabolite of the environmental carcinogen benzo[a]pyrene, predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. Here we developed an improved method for simultaneous analysis and purification of four stereoisomeric adducts in short oligonucleotides using reversed-phase high-performance liquid chromatography, providing a selection strategy of stationary phase for analysis and separation of polyaromatic hydrocarbon-DNA adducts. This work demonstrates that secondary retention of oligonucleotides on C18 stationary phases induced by exposed silanol heavily affects the separation of four stereoisomeric adducts on C18 stationary phases, and the silicone polymer monolayer coating for completely capping exposed silica or silanol greatly reduces such secondary retention, thereby displaying a much better resolution of the four stereoisomeric adducts. We further demonstrate that aromatic group (phenyl)-based stationary phase can significantly improve stereoisomeric separation of four anti-BPDE-deoxyguanosine (dG) adducts in short oligonucleotides over nonaromatic C18 stationary phase due to enhancement of the selective interaction with aromatic anti-BPDE moiety in oligonucleotides. The developed method was also used for purification and preparation of anti-BPDE-oligonucleotide adducts.     

Micelle-sensitized constant-energy synchronous fluorescence spectrometry for the simultaneous determination of pyrene, benzo[a]pyrene and perylene.

The use of micellar media in constant-energy synchronous fluorescence spectrometry has been proposed. The influence of some aqueous micellar systems on the determination of pyrene, perylene and benzo[a]pyrene has been investigated. The presence of these micellar systems allows their determination in aqueous media, thus avoiding the use of an organic solvent, and greatly enhances the fluorescence signals. The combination of a constant-energy synchronous scanning technique and a micellar system provided a single spectrum for the simultaneous identification and quantitative determination of the three polycyclic aromatic hydrocarbons (PAHs). Further there was no energy transfer among them, making the measurement simple and fast. A constant-energy difference of 2800 cm(-1) was selected. The analytical characteristics of the proposed method in the presence of anionic micelles of sodium dodecylsulfate (SDS) were studied. The detection limits were at a level of ng ml(-1). Analysis of water samples from two different origins spiked with known amount of pyrene, perylene and benzo[a]pyrene also gave satisfactory results, and total average recoveries were greater than 97.1%.     

Degradation of anthracene, pyrene and benzo[a]-anthracene in aqueous solution by chlorine dioxide

Polycyclic aromatic hydrocarbons (PAHs) constitute an important group of micropollutants, which are known to be mutagenic, carcinogenic and/or co-carcinogenic and relatively persistent in the environment. The effects of chlorine dioxide (ClO2) on the degradation of anthracene (ANTH), pyrene (PYR) and benzo[a]anthracene (BaA) in aqueous solution were investigated using high performance liquid chromatography (HPLC). In preliminary experiments, it was observed that ClO2 could remove these three PAHs effectively within a short time. Several factors including reaction time, the concentration of ClO2 and pH of the reaction mixture influencing the degradation ratio of PAHs have been studied by batch experiments. The results showed that the degradation ratio of PAHs was affected by reaction time and the concentration of ClO2 instead of pH. The degradation ratio of ANTH, PYR and BaA could reach their maximum as approximately 99.0%, 67.5% and 89.5%, respectively, under the condition as follows: reaction time 30, 60 and 120 min, the concentration of ClO2 0.1, 0.4 and 0.5 mmol·L-1, and pH 7.2. ANTH was selected as the representative to study the reaction mechanism with ClO2. The oxidation products formed in the reaction of ANTH with ClO2 were tentatively identified by gas chromatography-mass spectrometry (GC-MS), and the results showed that the main product was 9, 10-anthraquinone, which could be biodegraded more easily and quickly than ANTH. Through analyzing the reaction properties of ANTH and ClO2, the possible pathway for the ANTH-ClO2 reaction was proposed based on the theory of single electron transfer (SET).     

Separation and detection of a benzo[a]pyrene metabolite with capillary electrophoresis in the presence of DNA using laser-induced fluorescence

A novel approach was developed for the separation and detection of a benzo[a]pyrene B[a]P-DNA metabolite (tetrol I-1) in the presence of DNA using CE and laser induced fluorescence. Tetrol I-1 in the presence of DNA is intercalated and undergoes fluorescence quenching. Thus, an equilibrium is established between the intercalated tetrol I-1 and the uncomplexed tetrol I-1. It is only the uncomplexed tetrol that is fluorescent in the presence of DNA. The tetrol I-1 fluorescence intensities, at two concentrations of tetrol I-1 (0.005 and 0.01mg ml(-1)), were observed while varying the DNA concentration. Stern-Volmer plots were constructed of the fluorescence intensity of the uncomplexed tetrol I-1 versus DNA concentration. From the slopes of the Stern-Volmer plots quenching constants were determined. The quenching constants are essentially the same as an association constant for tetrol I-1 with DNA. The average value obtained for the association constant for the two concentrations of tetrol I-1 was 0.22+/-0.02 mg ml(-1). It was thus demonstrated that uncomplexed tetrol I-1 can be separated from DNA by CE and an association constant for tetrol I-1 bound to DNA can be obtained from the fluorescence quenching data.     

Superoxide chemical transformation of diolepoxide polyaromatic hydrocarbon DNA adducts. Determination of benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol by gas chromatography.

Benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol (100 pg, 342 fmol) was measured using the following sequence of steps: (1) chemical transformation with potassium superoxide to 2,3-pyrenedicarboxylic acid; (2) electrophore derivatization with pentafluorobenzyl bromide; (3) sample clean-up by high-performance liquid chromatography and (4) measurement by gas chromatography with electron-capture detection and by gas chromatography with electron-capture negative-ion mass spectrometry. The overall, absolute yields obtained by the two procedures were 69% and 60%, respectively. This work completes the first stage towards the establishment of a general method for detecting diolepoxide polyaromatic hydrocarbon DNA adducts by gas chromatography.