Arthur L. Lafleur

Human cell mutagenicity of oxygenated, nitrated and unsubstituted polycyclic aromatic hydrocarbons associated with urban aerosols

Polycyclic aromatic compounds (PAC) are ubiquitous pollutants in urban air that may pose risks to human health. In order to better assess the health risks associated with this class of compounds, a total of 67 PAC that either have been identified (55) or are suspected to be present (12) in urban aerosol samples were tested for mutagenicity in a forward mutation assay based on human B-lymphoblastoid cells. The cell line used (designated h1A1v2) constitutively expresses the cytochrome P4501A1, which is known to be necessary for the metabolism of many promutagens. The PAC tested included 39 polycyclic aromatic hydrocarbons (PAH). 19 oxygen-containing PAH (oxy-PAH) and nine NO2-substituted PAH (nitro-PAH). A total of 26 PAH were mutagenic. In comparing the minimum mutagenic concentrations of the mutagenic PAH with that of benzo[a]pyrene (B[a]P) it was found that dibenzo[a,l]pyrene (DB[al]P), cyclopenta[c,d]pyrene (CPP), naphtho[2,1-a]pyrene, dibenzo[a,e]pyrene (B[a]P) and 1-methylbenzo[a]pyrene were 24 +/- 21, 6.9 +/- 4.2, 3.2 + 3.0, 2.9 +/- 2.9 and 1.6+/- 1.4 times, respectively, more mutagenic than B[a]P, and that dibenzo[a,k]fluoranthene and B[a]P were approximately equally mutagenic. The 19 other mutagenic PAH were between approximately 2 and approximately 1800 times less mutagenic than B[a]P. Of the oxy-PAH tested only phenalenone, 7H-benz[d,e]anthracen-7-one, 3-nitro-6H-dibenzo[b,d]pyran-6-one, cyclopenta[c,d]pyren-3(4H)-one, 6H-benzo[c,d]pyren-6-one (BPK) and anthanthrenequinone were mutagenic; however, with the exception of BPK, these were over 50 times less active than B[a]P, BPK was approximately 3 times less active than B[a]P. Seven of the nitro-PAH were mutagenic including 9-nitroanthracene, 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,3-dinitropyrene, 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene. 1,6-DNP was approximately 4 times less active than B[a]P; the six other mutagenic nitro-PAH were between 20 and 380 times less active than B[a]P. These results are discussed in terms of their relevance for determining the most important mutagens in ambient air. Based on reported concentrations of PAC in ambient aerosols, it is possible that CPP, DB[ae]P, DB[al]P and BPK could account for a greater proportion of the mutagenicity than B[a]P in some aerosols.

Fullerenes synthesis in combustion

Abstract The early suggestion in fullerenes research that fullerenes might be produced in flames was soon supported by the observation of polyhedral carbon ions in flames and in 1991 was confirmed by the recovery and identification of fullerenes C60 and C70 from benzene/oxygen flames. Recent research has determined the effects of pressure, carbon/oxygen ratio, temperature and the type and concentration of diluent gas, on the yields of C60 and C70 in subatmospheric pressure premixed laminar flames of benzene and oxygen. Similar flames but with acetylene as fuel have also been found to produce fullerenes, but in smaller yields than with benzene fuel. The largest observed yields of C60 + C70 from benzene/oxygen flames are substantial, being 20% of the soot produced and 0.5% of the carbon fed. The largest rate of production of C60 + C70 was observed at a pressure of 69 Torr, a C/O ratio of 0.989 and a dilution of 25% helium. Several striking differences between fullerenes formation in flames as compared to the widely used graphite vaporization method include, in the case of flames, an ability to vary the C70/C60 ratio from 0.26 to 8.8 (cf., 0.02 to 0.18 for graphite vaporization) by adjustment of flame conditions and production of several isomers each of fullerenes C 60, C70, C60O and C70O. Many of the apparent isomers are thermally metastable, one C60 converting to the most stable form with a half-life of 1h at 111°C. The structures of the apparent C60 and C70 isomers necessarily must include abutting five-membered rings, previously assumed to be disallowed because of their high strain energy. The chemistry of fullerenes formation in flames is in some ways similar to that of soot formation, but important differences are seen and assumed to reflect the effects of the curved, strained structures of fullerenes and their precursors.

Isolation of fullerene products from flames: Structure and synthesis of the C60-cyclopentadiene adduct

Abstract A C 60 -C 5 H 6 adduct was isolated from a low pressure benzene flame. The structure of this product is identical to the Diels-Alder adduct of C 60 and cyclopentadiene as established through independent synthesis. This product was determined to be adduct 1 through 13 C and proton NMR, as well as infrared and mass spectrometry.

Characterization of flame-generated C10 to C160 polycyclic aromatic hydrocarbons by atmospheric-pressure chemical ionization mass spectrometry with liquid introduction via heated nebulizer interface

Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) generated from fuel-rich combustion of ethylene-naphthalene mixtures in a jet-stirred-plug-flow reactor were chemically characterized by combined mass spectrometric techniques to yield product composition data that cover the molecular mass region from simple PAHs (naphthalene, 128 u) to large molecules comparable in molecular size (1792 u) to nanoparticles of soot. Two techniques based on atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) were investigated: (1) APCI-MS combined with high-performance liquid chromatography through a heated nebulizer interface was found suitable for PAHs up to C36 (448 u). (2) For the characterization of larger PAHs beyond C36, direct liquid introduction (DLI) of sample into an atmospheric-pressure chemical ionization mass spectrometer through a heated nebulizer gave protonated molecular ions for PAHs over the m/z 400–2000 range. Although unequivocal elemental composition information is unattainable from the unit-resolution DLI/APCI-MS data, by starting with structural data from identified C16 to C32 PAHs, and applying PAH molecular growth principles, it was possible to generate PAH molecular maps from the DLI/APCI-MS data from which values for the elemental composition could be derived for all major peaks.

Multimode size exclusion chromatography with poly(divinylbenzene)/N-methylpyrrolidinone for the characterization of coal-derived mixtures

Abstract Multimode size exclusion chromatography (SEC) was investigated for the characterization of pyridine extracts of coal-derived materials. A system consisting of a poly(divinylbenzene) (PDVB) column with a mobile phase of N-methylpyrrolidinone (NMP) was evaluated. Model compounds covering a wide polarity range were studied to shed light on the separation mechanism. For neutral to moderately polar compounds, elution proceeded in order of decreasing molecular weight. Compounds showing predominantly size-dependent elution include polycyclic aromatic hydrocarbons, polyphenyls and azaarenes. For polar compounds, retention was largely independent of size effects and was governed mainly by polarity. In general, increasing polarity decreased the elution volume. Some polyfunctional polar compounds gave markedly decreased elution volumes compared with their neutral parent compounds. Increasing the extent of alkylation led to increased elution volumes for all compound types. Results are presented for pyridine extracts of Pittsburgh No. 8 bituminous, Montana Rosebud No. 10 subbituminous and Zap ND lignite. Chromatograms of raw coal extracts were characterized by a dominant, early-eluting peak consisting of highly-polar species whose retention was independent of size. The use of NMP as mobile phase permitted the dissolution and separation of highly polar material found in raw coal extracts. Also, it made possible the size-dependent separation of unsubstituted PAC, compounds that are abundant in extensively pyrolysed coal products.

Effects of PAH isomerizations on mutagenicity of combustion products

Abstract Most of the mutagenicity of mixtures of polycyclic aromatic hydrocarbons (PAH) mixtures found in combustion exhaust gases is contributed by a relatively small number of the many PAH present. Since PAH mutagenicity is structure and hence isomer sensitive, changes in the distribution of isomers can change the mutagenicity of the mixture. Whether isomerization reactions in combustion play a significant role in determining the distributions of PAH isomers and the mutagenicity of product mixtures is assessed here for the following pairs of isomers: 1. (1) fluoranthene-pyrene 2. (2) fluoranthene-acephenanthrylene 3. (3) cyclopenta[ cd ]pyrene-benzo[ ghi ]fluoranthene 4. (4) benzo[ k ]fluoranthene-benzo[ a ]pyrene Concentration ratios of the isomer pairs were measured in ethylene combustion with naphthalene injection using a plug flow reactor at equivalence ratios of 1.2 and 2.2 and temperatures of 1520, 1620, and 1705 K, and compared with equilibrium ratios based on properties computed from molecular mechanics and semiempirical quantum mechanical programs [MM3; MNDO, AM1, and PM3 in both restricted- and unrestricted-Hartree-Fock forms]. Bacterial mutagenicity was measured by a forward mutation assay using Salmonella in the presence of rat liver supernatant, and found to vary significantly among the above compounds. The measured concentration ratios for isomer pairs (2) and (3) are near the equilibrium values and becoming more so as temperature increases, but the measured ratios for isomer pairs (1) and (4) are far from the equilibrium values at all the temperatures. From kinetics estimations, the characteristic isomerization time for isomer pairs (2) and (3) at 1705 K and perhaps at 1620 K is less than the experimental residence times, while the only isomerization mechanisms envisioned for isomer pairs (1) and (4) would not be kinetically viable at these temperatures. These results indicate that isomerizations affecting mutagenicity are significant under these conditions for isomer pairs (2) and (3), which involve only compounds containing both five- and six-membered rings, but not for isomer pairs (1) and (4), each of which includes a compound containing only six- membered rings.

Mutagenicity of C24H14 PAH in human cells expressing CYP1A1.

Relatively little is known about the mutagenicity of C24H14 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 C24H14 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 C24H14 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 C24H14 PAH tested. These observations are discussed with emphasis given to identifying C24H14 PAH that may be important environmental mutagens.

The Atmosphere of Mars near the Surface: Isotope Ratios and Upper Limits on Noble Gases

Several new analyses of the martian atmosphere have been carried out with the mass spectrometer in the molecular analysis experiment. The ratios of abundant isotopes of carbon and oxygen are within 10 percent of terrestrial values, whereas nitrogen-15 is considerably enriched on Mars. We have detected argon-38 and set new limits on abundances of krypton and xenon. The limit on krypton is sufficiently low to suggest that the inventories of volatile substances on Mars and on Earth may be distinctly different.

Fullerenes and PAH in low-pressure premixed benzene/oxygen flames

The formation of fullerenes is though to be a molecular weight growth process similar to the formation of polycyclic aromatic hydrocarbon (PAH) and soot in flames, although little is known about the specific mechanisms involved. The goal of this study is to investigate possible fullerences formation pathways. This work measures for the First time concentration profiles of fullerences C60, C70, C76, C78, and C84, PAHs (up to 300 amu), and unidentified PAHs in the mass range between PAHs and soot in a heavily fullerene-forming premixed benzene/oxygen flame operated at the following conditions: fuel equivalence ratio, 2.4 (atomic C/O ratio, 0.96): cold gas velocity, 25 cm/s: pressure, 5.33 kPa: and fraction of argon in fuel mixture, 10 mol%. Two regions of fullerenes formation are identified in this flame. The First formation region occurs early in the flame simultaneously with PAH consumption. The rate of PAH consumption is more than large enough to account for the obsered rate of fullerenes formation, so this formation region may involve reactions of PAH. The Second region, which accounts for most of the fullerenes mass produced in the flame, occurs later in the flame and is more consistent with stepwise acetylene addtion to fullerene precursors. In both regions, fullerenes consumption that may involve reactions between fullerenes and soot is observed. In addition, measurements indicate that the different PAHs grow roughly simultaneously, reach a peak concentration, and decay together in a consumption process that occurs at the same time as a rapid rise in soot mass concentration early in the flame. This behavior is consistent with the major soot formation being from PAH and occurring along with the minor fullerenes formation in this flame.

Bacterial and human cell mutagenicity study of some C18H10 cyclopenta-fused polycyclic aromatic hydrocarbons associated with fossil fuels combustion.

A number of isomeric C18H10 polycyclic aromatic hydrocarbons (PAHs), thought to be primarily cyclopenta-fused PAHs, are produced during the combustion and pyrolysis of fossil fuels. To determine the importance of their contributions to the total mutagenic activity of combustion and pyrolysis samples in which they are found, we characterized reference quantities of four C18H10 CP-PAHs: benzo[ghi]fluoranthene (BF), cyclopenta[cd]pyrene (CPP), cyclopent[hi]acephenanthrylene (CPAP), and cyclopent[hi]aceanthrylene (CPAA). Synthesis of CPAA and CPAP is described. The availability of reference samples of these isomers also proved to be an essential aid in the identification of the C18H10 species often found in combustion and pyrolysis samples. Chemical analysis of selected combustion and pyrolysis samples showed that CPP was generally the most abundant C18H10 isomer, followed by CPAP and BF. CPAA was detected only in pyrolysis products from pure PAHs. We tested the four C18H10 PAHs for mutagenicity in a forward mutation assay using S. typhimurium. CPP, BF, and CPAA were roughly twice as mutagenic as benzo[a]pyrene (BaP), whereas CPAP was only slightly active. These PAHs were also tested for mutagenic activity in human cells. In this assay, CPP and CPAA were strongly mutagenic but less active than BaP, whereas CPAP and BF were inactive at the dose levels tested. Also, the bacterial and human cell mutagenicity of CPAA and CPAP were compared with the mutagenicity of their monocyclopenta-fused analogs, aceanthrylene and acephenanthyrlene. Although the mutagenicities of CPAP and acephenanthrylene are similar, the mutagenic activity of CPAA is an order of magnitude greater than that of aceanthyrlene.