Richard A. Pelroy

Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.

Identification of primary aromatic amines in mutagenically active subfractions from coal liquefaction materials.

Gas-chromatographic mass spectral (GCMS) analyses were performed on mutagenically active components from the basic, basic tar and neutral tar subfractions of a coal-derived liquid heavy distillate. The latter material is a component fraction of an experimental oil produced in a solvent refined coal process (SRC II) pilot plant. Mutagenicity was determined with the Ames/Salmonella assay system. Thin-layer chromatography (TLC) was used to separate mutagenically active components of the basic, basic tar and neutral tar fractions from some of the other compounds contained in these complex materials. For the 3 fractions tested, mutagenic activity was localized in approx. the same TLC regions, with relative mobilities (Rf) ranging from 0.1 to 0.3. GCMS analysis of the TLC regions showed that the concentrations of primary aromatic amines, as obtained by measuring peak areas for the (M + 1)+ ion as formed in the chemical ionization mode, followed essentially the same distribution as the mutagenic activity. Primary aromatic amines identified in the active regions included aminonaphthalenes, aminoanthracenes, aminophenanthrenes, aminopyrenes and aminochrysenes. With the exception of small amounts of aminonaphthalene, primary aromatic amines were not found in TLC regions that lacked mutagenic activity.

In-vitro assay for mutagenic activity and gas chromatographic-mass spectral analysis of coal liquefaction material and the products resulting from its hydrogenation

Abstract Employing assays for microbial mutagenesis activity together with solvent fractionation and gas chromatographic-mass spectral analysis, coal liquefaction material was compared to its catalytically hydrogenated products with regard to biological activity and chemical composition. The mutagenic activity of the moderate and severly hydrogenated products was substantially reduced compared to that of the feedstock. In contrast to the essentially aromatic nature of the principal components in the coal oil feedstock, the hydrogenated products were shown to contain mainly hydroaromatic species with a significant decrease in the fraction of basic and tar components as obtained by our acid-base solvent separation procedure. Subfractions of the basic and tar components obtained from the feedstock were shown to contain primary aromatic amines, the concentrations of which were essentially in direct proportion to the observed microbial mutagenic activity. No primary aromatic amines were detected in the hydrogenated products. Hydrotreating appears to produce a coalderived liquid with far less mutagenic activity than the untreated coal liquefaction material.

Comparative chemical composition and biological activity of single- and two-stage coal liquefaction process streams

Abstract Samples from several integrated two-stage coal liquefaction (ITSL) process streams were collected under both normal and off-normal operating conditions. Selected crude materials were tested for their ability to initiate tumorigenicity in the initiation/promotion assay in mouse skin. Crude materials and their chemical class fractions were assayed for microbial mutagenic activity in the standard Ames test with S. typhimurium TA98, and selected samples were also subjected to the Chinese hamster ovary mammalian cell assay for mutagenicity. Mass spectral and gas chromatographic—mass spectral (g.c.—m.s.) analyses were carried out on these materials. Results were compared with those for analogous materials from solvent refined coal (SRC) single stage liquefaction processes. In general, the ITSL distillates were of higher molecular weight, higher hydroaromatic content, lower nitrogen content and were somewhat less alkylated than the analogous SRC materials. Compared to the SRC coal liquefaction materials, the ITSL distillates and bottoms materials collected under normal run conditions were substantially less active in both the microbial and mammalian cell mutagenicity assays. These same materials were also less active than ITSL samples collected under off-normal conditions. G.c.—m.s. analyses showed that ITSL materials collected under normal operating conditions were substantially reduced in amino polycyclic aromatic hydrocarbon content when compared to both the off-normal ITSL, and SRC materials. Activity of the ITSL materials as initiators of tumorigenesis was greater than expected, based on results from the in vitro assays and not significantly different than that of the SRC materials. The higher initial boiling point of the ITSL distillates probably accounts for much of their greater than expected initiating activity compared to the SRC materials.

Mutagenic characterization of synthetic fuel materials by the Ames/Salmonella assay system☆

Solvent refined coal (SRC) distillates produced by 2 different pilot plant processes were compared for mutagenicity using the Ames/salmonella assay. The high-molecular-weight, high-boiling SRC distillates were fractionated into their various constituent chemical classes for Ames analysis. In each case, the major portion of the mutagenic activity contained in the crude materials was recovered in the basic and in the tar fractions. Little or no activity was found in the neutral. aliphatic, or neutral polyaromatic hydrocarbon (PAH)-enriched fractions. The most active of the SRC subfractions were comparable in mutagenicity to benzo[a]pyrene, but substantially less active than 2-aminoanthracene.

Relative concentrations of polyaromatic primary amines and azaarenes in mutagenically active nitrogen fractions from a coal liquid

Thin-layer chromatography (TLC) was used to separate components in the basic and tar fractions of solvent refined coal (SRC-I) process solvent (PS) to obtain materials suitable for biological and chemical analysis. Those fractions eluted from TLC plates which were mutagenically active in the Ames/Salmonella assay were analyzed by gas chromatographic mass spectrometry (GCMS) for polycyclic azaarenes, polyaromatic primary amines (PAA) and carbazoles. In all materials tested, a strong correlation was observed between the concentration of PAAs in a given TLC region and the mutagenicity of that region in the Ames assay system. Conversely, azaarenes having 2--4 fused rings and carbazoles were present in both mutagenic and non-mutagenic TLC eluates. No PAAs were detected in mutagenically inactive TLC eluates. In comparison to the mutagenic tar fractions, the PS basic fraction contained relatively larger concentrations of 2- and 3-ringed components such as aminonaphthalenes and aminoanthracenes or aminophenanthrenes. The tar fractions, which were essentially devoid of aminonaphthalenes, had a higher average molecular weight and contained relatively higher concentrations of aminopyrenes.

Comparison of the mutagenicities of fossil fuels

Chemical fractions from 4 shale oils including one produced in an above-ground retort (Paraho), 2 produced below ground by in situ method (Occidental and Geokinetics), and 1 produced by a simulated modified in situ process (Livermore), were tested for mutagenicity against Salmonella typhimurium TA98 in the Ames assay system. Their mutagenic activities were compared with 2 other complex materials: a high-boiling coal distillate (The Solvent Refined Coal Process I (SRC II) heavy distillate), and a crude petroleum (Prudhoe Bay). Each material was fractionated by Sephadex LH-20 partition chromatography and by acid-base solvent extraction to provide chemical fractions for bioassay. Mutagenic activity was detected in the coal liquid (and their fractions), but not in the natural crude oil nor in any of its fractions. The basic and tar fractions derived by solvent extraction, and the LH-20 methanol fractions contained most of the mutagenic activity recovered from the unfractionated material. The heavy distillate was approx. 10 times more active in the Ames assay than the most mutagenically active shale oil. The most mutagenically active of the shale oils was about 10 times more active than the least active shale oil.

The effects of nitrous acid on the mutagenicity of two coal liquids and their genetically active chemical fractions

The Ames/Salmonella assay was used to determine the effect of nitrous acid on the mutagenicity of solvent refined coal (SRC) distillates and distillate fractions. The SRC materials consisted of the higher-molecular-weight, high-boiling distillates, the process solvent (PS) and heavy distillate (HD), and also included the basic tar and neutral tar chemical fractions derived from these 2 distillates. Nitrosation products of each of the SRC materials were less mutagenic than the distillates of distillate fractions from which they were derived (untreated materials); in most cases they were less than 10% as mutagenic as that observed in the starting materials. The mutagenicity of the SRC materials after nitrous acid treatment was direct-acting, whereas all mutagenicity associated with the untreated SRC materials was indirect and required metabolic activation for expression in the Ames assay system.

Partition chromatography-high-performance liquid chromatography facilitates the organic analysis and biotesting of synfuels

Abstract Partition chromatography (Sephadex LH-20 or C 18 -partition) followed by high-performance liquid chromatography facilitated the organic and mutagenic characterization of synfuel samples. The mutagens in oil shale retort waters were polar, whereas those in a shale oil ranged from moderately polar to polar. In contrast, the mutagens in an solvent refined coal-II (SRC-II) distillate blend were primarily moderately polar. The mutagens in an SRC-I process solvent were both less polar and less heterogeneous than those of an SRC-I solid product. Known mutagens (primary aromatic amines and aza-polynuclear aromatic hydrocarbons) were identified in the SRC-I and SRC-II samples but not in the shale oil and retort waters.

Effect of process distillation on mutagenicity and cell-transformation activity of solvent-refined, coal-derived liquids

Abstract Blended SRC-II process streams, representing a full boiling range distillate material, were fractionally distilled into non-overlapping 50 °F cuts with boiling points between 300 and 850 °F. Another set of 18 distillate cuts were obtained with boiling points ranging between 138 and 1055 °F. Distillate cuts were assayed for mutagenic activity using the histidine reversion assay with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as for mammalian-cell transformation activity in the Syrian hamster embryo test, and DNA damage in the prophage induction assay. Samples were also separated into chemical class fractions by alumina column chromatography and analysed by high resolution gas chromatography so that the chemical composition of the cuts could be related to their relative activity in the different assays. In the mammalian cell transformation and microbial mutagenicity assays, significant activity was found almost exclusively in distillate cuts with components boiling > 700 °F, with the highest activity in the transformation assay observed for cuts > 800 °F. All of the distillate cuts showed increased levels of DNA damage as expressed by lambda prophage induction in Escherichia coli 8177. However, the greatest activity was associated with distillate cuts with boiling points in the 800 °F + range. Chemical analysis of the 50 °F distillate cuts showed a variety of polycyclic aromatic hydrocarbons (PAH) and amino-PAH compounds to be present in the distillate cuts boiling > 700 °F and essentially absent from cuts boiling