Pyrene Isomers Research Articles

Capillary Electrochromatography Coupled to Atmospheric Pressure Photoionization Mass Spectrometry for Methylated Benzo[a]pyrene Isomers

Benzo[a]pyrene, one of the most carcinogenic PAHs, has 12 monomethylated positional isomers (MBAPs). A strong correlation between the carcinogenicity of these isomers and methyl substitution has been reported. In this study, on-line coupling of capillary electrochromatography (CEC) and atmospheric pressure photoionization mass spectrometry (APPI-MS) provides a unique solution to highly selective separation and sensitive detection of MBAP isomers. The studies indicated that APPI provides significantly better sensitivity compared to electrospray ionization and atmospheric pressure chemical ionization modes of MS. A systematic investigation of APPI-MS detection parameters and CEC separation is established. First, several sheath liquid parameters (including type and concentration of volatile buffers, type and content of organic modifiers, use of dopants and inorganic/organic additives, and sheath liquid flow rate) and APPI-MS spray chamber parameters (capillary voltage, vaporizer temperature, nebulizer pressure) were found to have effects on detection sensitivity as well as the profile of mass spectrum. For example, when ammonium acetate was replaced with acetic acid in the sheath liquid, the MS signal was enhanced as much as 90% and the formation of ammonia adduct was effectively suppressed. Next, the separation of MBAP isomers was conducted on internal tapered columns packed with polymeric C18 stationary phase. With the use of a mobile phase consisting of slightly higher acetonitrile content (90%,v/v) and a small amount of tropylium ion, the analysis times were significantly shortened by 20 min without compromising the resolutions between the isomers. Finally, quantitative aspects of the CEC-APPI-MS method were demonstrated using 7-MBAP as the internal standard. The calibration curves of three of the most carcinogenic isomers, namely, 1-MBAP, 3-MBAP, and 11-MBAP, showed good linearity in the range of 2.5-50 microg/mL with a limit of detection at 400 ng/mL.

Error-prone Translesion Synthesis by Human DNA Polymerase η on DNA-containing Deoxyadenosine Adducts of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.

Reference range levels of polycyclic aromatic hydrocarbons in the US population by measurement of urinary monohydroxy metabolites

We developed a gas chromatography isotope-dilution high-resolution mass spectrometry (GC/ID-HRMS) method for measuring 14 polycyclic aromatic hydrocarbon (PAH) metabolites representing seven parent PAHs in 3 mL of urine at low parts-per-trillion levels. PAH levels were determined in urine samples collected in 1999 and 2000 from approximately 2400 participants in the National Health and Nutrition Examination Survey, and, for the first time, reference range values were calculated for these metabolites in the US population. Using this GC/ID-HRMS method, we found detectable concentrations for monohydroxy metabolite isomers of fluorene, phenanthrene, fluoranthene, pyrene, and chrysene, benzo[c]phenanthrene, and benz[a]anthracene. Some monohydroxy metabolite isomers of benzo[c]phenanthrene, chrysene, and benz[a]anthracene exhibited low detection frequencies that did not allow for geometric mean calculations. Our study results enabled us to establish a reference range for the targeted PAHs in the general US population.

Theoretical (DFT, GIAO–NMR, NICS) study of carbocations (M+H)+, dications (M2+) and dianions (M2−) from dihydro-dicyclopenta[ef,kl]heptalene (dihydro-azupyrene), dihydro-dicyclohepta[ed,gh]pentalene, and related bridged [14]annulenes

The annulenium ions of protonation, the two-electron oxidation dications, and the two-electron reduction dianions derived from dihydro- and dimethyldihydro derivatives (cis and trans) of dicyclopenta[ef,kl]heptalene (azupyrene) (1) and dicyclohepta[ed,gh]pentalene (2), which are the nonalternant isomers of pyrene, were studied by density functional theory (DFT) at the B3LYP/6-31G(d), 6-31+G(d,p), or 6-31++G(d,p) levels. Charge delocalization modes in the energetically most favored annulenium ions, as well as in the singlet and triplet dications and dianions, were assessed based on gauge-including atomic orbital (GIAO) Deltadelta](13)C values and via changes in natural population analysis (NPA) charges. Relative aromaticity/antiaromaticity in the annulenes were gauged via nucleus-independent chemical shift (NICS) and DeltaNICS. Annulenium ions of monoprotonation, the dications, and dianions derived from bismethano- and propanediylidene [14]annulenes were also studied by DFT for comparison with the cis-dihydro isomers derived from . Computed GIAO NMR data and the optimized geometries were compared with the experimental data when available, and the optimized geometries were compared with the X-ray data if known. A basis-set dependency study of the computed GIAO chemical shifts was also undertaken. The present DFT work represents the first detailed comparative theoretical study of charged annulenes derived from the dihydro derivatives of and .

Capillary electrochromatography of methylated benzo[a]pyrene isomers: II. Effect of stationary phase tuning

For Part II of our ongoing study, we present a strategy for stationary phase optimization for the capillary electrochromatographic (CEC) separation of the 12 methylated benzo[a]pyrene (MBAP) isomers. Utilizing the optimum mobile phase conditions from Part I of our study as a guide, seven commercially available stationary phases have been evaluated for their ability to separate highly hydrophobic MBAP isomers. Ranging in design from high-performance liquid chromatography (HPLC) to CEC application, each phase was slurry packed in house and tested for CEC suitability and performance. Several stationary phase parameters were investigated for their effects on MBAP separation including bonding type (monomeric or polymeric, % carbon loading, surface coverage), pore size, particle size, and type of alkyl substituent. In this manner, the present state of commercially available packings has been assessed in our laboratory. Utilizing the optimum polymeric C 18-5 μm-100 Å-PAH stationary phase, the effects of CEC packed bed length and capillary inside diameter (I.D.) were also evaluated. A 50 μm I.D. capillary, 25 cm packed bed length and 75% (v/v) acetonitrile, 12.5 m M Tris, pH 8.0, 20 °C at 30 kV, provided resolution of 11 out of 12 MBAP isomers thus showing the effectiveness of CEC for analysis of structurally similar methylated polyaromatic hydrocarbons.

Capillary electrochromatography of methylated benzo[ a]pyrene isomers : I. Effect of mobile phase tuning

It is of increasing importance that chromatographic methods be developed for the separation and identification of biological and environmentally harmful compounds such as methylated polycyclic aromatic hydrocarbons (PAH). Capillary electrochromatography (CEC) is fast becoming a useful technique for analysis of PAH, as it offers both high efficiency and superior resolution. The separation of 12 methylated benzo[ a]pyrene (MBAP) isomers is a challenge due to the extreme hydrophobicity and structural similarity of these compounds. In this work, we present Part I of our ongoing study, a method for the systematic mobile phase tuning for CEC separation of the 12 MBAP isomers. The CEC experiments were conducted utilizing a CEC-octadecylsilica (ODS) stationary phase and fused-silica capillary [(75 μm I.D., 363 μm O.D.) 36.5 cm total length, 25.0 cm effective length] which was slurry pressure packed in our laboratory. Several mobile phase parameters were manipulated to provide optimum separation. These included acetonitrile (ACN) concentration, tris(hydroxymethyl) aminomethane (Tris) concentration, pH, and addition of a tertiary buffer constituent such as tetrahydrofuran (THF) and isopropanol (IPA) to ACN–aqueous buffer mixtures. Optimum CEC separation conditions were achieved using 75% (v/v) ACN–25% (v/v) 12.5 m M Tris, pH 8.0, and 30 kV at 25 °C. These mobile phase conditions were then utilized for Part II of our study, the CEC stationary phase optimization for the separation of 12 MBAP isomers.

Method for determining monohydroxybenzo[ a]pyrene isomers using column-switching high-performance liquid chromatography

A method for determining monohydroxybenzo[ a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a β-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3–8 pg/injection ( S/ N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[ a]pyrene in vitro by human recombinant P450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by β-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91–103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.

High-energy collision-induced dissociation of small polycyclic aromatic hydrocarbons.

High-energy collision-induced dissociation (CID) experiments on polycyclic aromatic hydrocarbons (PAHs) having 2-6 rings, naphthalene, anthracene, phenanthrene, fluoranthene, pyrene and coronene, were performed, and the relative abundances of their fragment ions were investigated as a function of collision energy. The results revealed that the PAHs except naphthalene showed a bimodal-type distribution of positive fragmentation ions, which is closely similar to the fragment-ion distribution reported for the CID of three-dimensional fullerene, C(60)(+) and C(70)(+). The three-ring isomers of anthracene and phenanthrene and the four-ring isomers of fluoranthene and pyrene can be distinguishable in their spectra under an electron ionization energy of 70 eV, but the high-energy CID spectra of the three- and four-ring isomers were almost identical. The fragmentation corresponding to fragment ions in the low-mass region of the bimodal CID spectra could be interpreted by the simple statistical model that fragment ions are formed by random evaporation from the molecular ions after a considerable structural rearrangement, 'phase transition', occurring at some high-energy state.

Micellar electrokinetic chromatography of monohydroxybenzo[a]pyrene positional isomers using γ-cyclodextrin

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the separation of benzo[a]pyrene and 12 positional isomers of monohydroxybenzo[a]pyrenes using γ-cyclodextrin together with sodium dodecyl sulfate. The optimum running conditions were found to be 20 mM phosphate–5 mM borate buffer (pH 8.5) containing 20 mM γ-cyclodextrin and 50 mM sodium dodecyl sulfate with an effective voltage of 12 kV at 20 °C. Octanol–water distribution coefficients of the compounds in the presence of γ-cyclodextrin were measured as an index of the association constants for complexes of γ-cyclodextrin with these compounds. The migration time of the analytes correlated well with their distribution coefficients, suggesting that their migration order could be correctly predicted on the basis of calculations of host–guest interaction energies. The relationship between the migration order and the structures of monohydroxybenzo[a]pyrene isomers is discussed.

Mutagenicity of C 24H 14 PAH in human cells expressing CYP1A1

Relatively little is known about the mutagenicity of C 24H 14 PAH, a diverse group of five- and six-ring PAH, some of which are present at trace levels in the environment. To better understand the mutagenicity of this class of compounds, 11 C 24H 14 PAH, including benzo[ a]perylene, benzo[ b]perylene, dibenzo[ a, e]fluoranthene, dibenzo[ a, f]fluoranthene, dibenzo[ j, l]fluoranthene, dibenzo[ a, h]pyrene, dibenzo[ a, i]pyrene, dibenzo[ e, l]pyrene, naphtho[1,2- b]fluoranthene, naphtho[2,3- a]pyrene, and naphtho[2,3- e]pyrene, were tested in a mutagenicity assay based on human h1A1v2 cells. h1A1v2 cells are a line of human B-lymphoblastoid cells that have been engineered to express cytochrome P4501A1 (CYP1A1), an enzyme capable of metabolizing promutagenic PAH. Mutagenicity was measured at the thymidine kinase ( tk) locus following a 72-h exposure period. Our results show that nine of the compounds were mutagenic. Benzo[ a]perylene, dibenzo[ a, e]fluoranthene, dibenzo[ a, i]pyrene, and naphtho[2,3- a]pyrene were the most potent mutagens, having minimum mutagenic concentrations (MMC) (i.e., the dose at which the induced response was twice that of the negative controls) in the 1–5 ng/ml range. Benzo[ b]perylene, dibenzo[ a, h]pyrene, dibenzo[ a, f]fluoranthene, and naphtho[2,3- e]pyrene were somewhat less potent mutagens, having MMC in the 10–30 ng/ml range. Dibenzo[ e, l]pyrene, which had an MMC of 280 ng/ml, was the least potent mutagen. Dibenzo[ j, l]fluoranthene and naphtho[1,2- b]fluoranthene were not mutagenic at the doses tested (1–3000 ng/ml). The most mutagenic compounds were also quite toxic. At the highest doses tested, benzo[ a]perylene, dibenzo[ a, e]fluoranthene, dibenzo[ a, i]pyrene, dibenzo[ a, h]pyrene, and dibenzo[ a, f]fluoranthene induced >60% killing, and naphtho[2,3- a]pyrene and naphtho[2,3- e]pyrene induced >50% killing. Benzo[ b]perylene, dibenzo[ e, l]pyrene, dibenzo[ j, l]fluoranthene, and naphtho[1,2- b]fluoranthene induced <50% killing at the highest doses tested. Comparing these results to a previous study in which nine other C 24H 14 PAH were tested for mutagenicity in this same assay, it was found that dibenzo[ a]pyrene isomers were generally more mutagenic than the other groups of C 24H 14 PAH tested. These observations are discussed with emphasis given to identifying C 24H 14 PAH that may be important environmental mutagens.

The Liquid Chromatographic Separation of Metabolites of Benzo[a]pyrene with γ-Cyclodextrin as a Mobile Phase Additive

Abstract The effects of γ-cyclodextrin as a mobile phase additive on the retention characteristics of fourteen metabolites of benzo[a]pyrene were investigated with reversed-phase liquid chromatography. It was possible to separate dihydrodiols, diones, and monohydroxyl-benzo[a]pyrenes as individual classes of metabolites. Also, with γ-cyclodextrin in the mobile phase, monohydroxyl-benzo[a]pyrene isomers that were very difficult to separate were readily separated. For example, 6-hydroxyl-benzo- [a]pyrene was separated from 12-hydroxyl-benzo[a]pyrene and 9-hydroxyl-benzo[a]pyrene was separated from 2-hydroxyl-benzo- [a]pyrene. The four tetrols were readily separated with shorter retention times using 4.0 mM of γ-cyclodextrin in a methanol-water (55:45) mobile phase. Overall, the results showed that γ-cyclodextrin was more effective in separating the metabolites than was β-cyclodextrin.

Reductive Transformations, 16[1] Electron Transfer and Regioselectivity in the Alkylation of a Hydrocarbon Mono‐ and Dianion

AbstractThe reductive alkylation of the pyrene isomer 1 affords bridged [14]annulenes (2) and 2a,3‐dihydro‐2a,3‐dialkyldicyclopenta[ef,kl]heptalenes (3). The involvement of an electron transfer (ET) in this reaction is demonstrated by optically pure electrophiles. Supporting evidence for ET processes is provided by cyclic voltammetric measurement of rate constants for both the ET process and the SN2 reaction. The choice of the electrophile is relevant not only for the importance of electron transfer, but also for the regioselectivity of the alkylation reaction.

Cyclopenta[cd] pyrene and monomethyl isomers

Cyclopenta[cd] pyrene and monomethyl isomers

Reductive transformations-14. Regioselective addition of carbanions to the “aromatic” pyrene isomer dicyclopenta(ef,kl)heptalene

We report on a LUMO-controlled nucleophilic addition to the pyrene isomer 1 . Treatment of 1 with organometallic reagents and subsequent quenching of the resulting monoanionic intermediates yield bridged [14]annulenes.

Determination of methylbenzo[a]pyrene isomers in a coal tar standard reference material using liquid chromatography and Shpol'skii spectrometry

Concentrations of individual methylbenzo[a]pyrene (MBP) isomers were determined in a coal tar standard reference material (SRM 1597) by using liquid chromatography (LC) (normal- and reversed-phase) and high resolution Shpol'skii spectrometry (HRS) at 15 K. This is the first report on the unambiguous identification and quantification of each MBP isomer in a real sample, and this report contains information on the distribution of these highly carcinogenic compounds in coal tar.

Epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene via a hydroperoxide-dependent mechanism catalyzed by lipoxygenases

The lipoxygenase catalyzed epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was examined. Epoxidation of the BP-7,8-diol was catalyzed by 5- and 15-lipoxygenase in the presence of either arachidonic acid, gamma-linolenic acid, or 15-hydroperoxyeicosatetraenoic acid (15-HPETE). The anti-9,10-epoxy-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene isomer was formed in greater quantities than the syn isomer, indicative of peroxyl radical mediated epoxidation. Epoxidation was dependent on time, enzyme and fatty acid concentration. There was no difference in the time course of epoxidation with either arachidonic acid or 15-HPETE, although the initial rate of oxygen consumption was approximately 55-fold greater with arachidonic acid. The lipoxygenase inhibitor and anti-oxidant nordihydroguaiaretic acid inhibited epoxidation in a dose-dependent manner in incubations initiated with either arachidonic acid or 15-HPETE. The anti-oxidant butylated hydroxyanisole also inhibited the epoxidation. Incubations conducted under anaerobic conditions with 15-lipoxygenase and either arachidonic acid or 15-HPETE significantly decreased epoxidation. This suggests that the oxygen inserted into BP-7,8-diol is derived from the atmosphere. The epoxidizing peroxyl radicals could not be detected but their precursors, carbon-centered radicals, were detected by using the ESR spin trapping technique in incubations of 15-lipoxygenase with 15-HPETE. This radical, formed by reduction and rearrangement of the hydroperoxide, may trap oxygen to form a peroxyl radical. We propose that the epoxidizing species is a peroxyl radical derived from 15-HPETE rather than from arachidonic acid. This proposal is based on the similar amounts of epoxidation, but dissimilar amount of oxygen consumed with both fatty acids. Since lipoxygenases are widely distributed in vivo, especially in areas where tumors arise such as the pulmonary epithelium, peroxyl radical formation by these enzymes may have an important role in chemical carcinogenesis.

Mutagenic activity of nitro-substituted cyclopenta-fused polycyclic aromatic hydrocarbons towards Salmonella typhimurium

Cyclopenta-fused isomers of pyrene and benz[ a]anthracene, nitrated on the etheno bridge, were synthesized and tested in the Ames plate-incorporation assay. Since enzymatic reduction, if it occurs in these compounds, would form arylhydroxylamines which in turn would form highly stabilized arylnitrenium ions, we hoped to test the hypothesis that the direct-acting mutagenic activity of nitroPAH is correlated with the degree of stabilization of the electrophilic intermediate. We found that these compounds are mutagenic (1–9 rev/nmole in Salmonella typhimurium TA98) and do not require S9 activation. However, this activity is substantially lower than that of other nitroPAH of comparable size such as 1-nitropyrene (250–300 rev/nmole). The reasons for this comparative lack of activity are discussed with reference to current theories regarding structure-activity relationships of nitroPAH.

Determination of 2-nitrofluoranthene and 2-nitropyrene in ambient particulate organic matter: Evidence for atmospheric reactions

We have identified and quantified 2-nitrofluoranthene (2-NO 2-FL) and 2-nitropyrene (2-NO 2-PY), both strong, direct mutagens in paniculate organic matter (POM) samples collected in polluted ambient air in southern California. Samples were collected during four consecutive 6-h periods on 18–19 September 1984, during which the ambient concentrations of gaseous co-pollutants were characterized by long pathlength spectroscopic techniques and conventional analyzers. Concentrations ranged up to 0.3 ng m −3 for 2-NO 2-FL and 0.02 ng m −3 for 2-NO 2-PY. The 2-nitro isomers of fluoranthene and pyrene have not been observed in direct emissions of POM (e.g. diesel exhaust and wood smoke). However, we recently observed these isomers from laboratory reactions involving N 2O 5 or OH radicals and the parent compounds. Thus the identification of 2-NO 2-FL and 2-NO 2-PY in ambient POM suggests that chemical transformations of fluoranthene and pyrene may take place in polluted atmospheres.

Thermal Deoxygenation of syn‐15, 16‐Dioxo‐1,6 : 8,13‐bismethano[14]annulene to the Pyrene Isomer Dicyclohepta[cd,gh]pentalene (Azuleno[2,1,8‐ija]azulene)

A unique reaction takes place in the pyrolysis of the diketone 1. The formation of the pyrene isomer 2 (60%) can hardly be explained other than in terms of via a 1,2-dioxetane; thus, this reaction would be the reverse of the oxidative cleavage of electron-rich double bonds with singlet oxygen. In addition, 30% anthracene is obtained by the expected stepwise decomposition of 1 via a norcaradienone derivative.

Chromatographic separation of benzo(a)pyrene isomers on nematic liquid crystal GC and polymeric reversed-phase HPLC phases

The use of two nematic liquid crystals (BABT and BPhBT) as GC stationary phases for the separation of monohydroxybenzo(a)pyrenes. as their TMS ethers, and monomethylbenzo(a)pyrenes was developed and compared with the separation of these isomers by HPLC using a polymeric ODS reversed-phase column. It was found that while HPLC and GC gave comparable separation of the hydroxy isomers, 10 out of 12 separated, better separation of the methyl isomers was obtained using HPLC. A simultaneous use of both HPLC and GC would resolve the twelve hydroxy isomers in about 70min. The results indicated that HPLC, using polymeric reversed-phase columns, is as powerful a tool as GC using nematic liquid crystal phases, for the separation of benzo(a)pyrene isomers. A discussion of the effect of solute length-to-breadth ratio on elution order is presented.