Comparative chemical and biological analysis of coal tar-based therapeutic agents to other coal-derived materials.

In this study, methodologies developed for the analysis of synthetic fuel products were applied to the coal tar fractions isolated from coal tar-based pharmaceutical products. A pharmaceutical stock solution of 20% coal tar in alcohol, a 50% coal tar bath emulsion and a 4.3% coal tar shampoo were studied. The toxicology and chemical composition of the coal tar fractions isolated from these materials were compared with an industrial coal tar and with a direct-liquefaction coal liquid product. The coal tars and coal liquid product were fractionated into chemical classes by alumina column chromatography and individual components were identified and quantitated by high-resolution gas chromatography. The microbial mutagenicity of these materials was measured against S. typhimurium, TA 98. In addition, the industrial coal tar, coal-liquid product, and coal tar isolate from the 20% coal tar in alcohol solution were tested for initiating activity in an initiation/promotion mouse skin painting assay for carcinogenicity. The chemical compositions of the coal tar-based therapeutic agents, the industrial coal tar and direct-liquefaction coal liquid were similar. With the exception of the 50% bath emulsion, the microbial mutagenicity and tumor-initiating activity in mouse skin for those materials tested were also similar.

Variation of composition with particle size in coal liquid aerosols generated for inhalation toxicology studies.

The chemical composition and microbial mutagenicity of aerosols generated by nebulizing two coal oils (solvent refined coal [SRC]-I process solvent [PS] and SRC-II heavy distillate) were found to vary with particle size. Significant quantities of the most volatile components of PS were also present as vapors. Evaporation and condensation processes in oil deposited on surfaces as well as in the aerosol are believed to be important in determining the observed composition changes. Complete physical and chemical characterization of the aerosol should be included in inhalation studies of complex materials since the animals may be exposed to material of quite different composition than that placed in the generator initially.

Microbial mutagenicity of isomeric two-, three-, and four-ring amino polycyclic aromatic hydrocarbons.

The isomers of various two-, three-, and four-ring amino polycyclic aromatic hydrocarbons were tested for mutagenic activity using a microbial plate incorporation test with four Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537). All compounds were assayed with an S9 metabolic activating enzyme system. The two-ring compounds were tested only with TA98. All were weakly mutagenic (1-10 rev/micrograms) except 2-aminobiphenyl, which was not mutagenic under these test conditions. All except two of the 13 fused three-ring compounds (aminofluorenes, aminoanthracenes, and aminophenanthrenes) were active frame shift mutagens; only the aminophenanthrenes were active base-pair mutagens. The potency of this group of isomeric compounds ranged from moderately (approximately 20 rev/microgram) to strongly (greater than 5,000 rev/microgram) mutagenic. As a group, the pericondensed four-ring amino compounds were the most mutagenic of the three groups tested. All of the aminofluoranthene and aminopyrene isomers showed significant mutagenic activity with TA98, TA100, and TA1537. In general, the mutagenic potency of the amino polycyclic aromatic compounds tested was highly dependent on the structural position of the amino group.

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.

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.

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.

Use of Ames test in evaluation of shale oil fractions.

Conditions that affect the sensitivity of the Ames assay of complex hydrocarbon mixtures derived from shale oil were studied. Two fractions, one enriched in polynuclear aromatic compounds (PNA fraction), and a second fraction enriched in aromatic and heterocyclic amines (basic fraction), were selected for most of this work because of their comparatively high mutagenicity (i.e., compared with raw shale oil). The crude shale oil, as well as the basic, PNA, and tar fractions were mutagenic against the Salmonella typhimurium test strains, TA98 and TA100. Mutation was dependent on metabolic activation by microsomal (S9) enzymes. Both test strains responded equally well to the crude product and to the basic fraction; however, strain TA100 was more effective than TA198 in demonstrating the mutagenicity of the PNA fraction. The mutagenicity of the tar fraction could be most easily detected after metabolic activation in a liquid medium, as opposed to S9 activation in the top agar of the standard Ames assay. The mutagenicity of the basic fraction or 2-aminoanthracene was also demonstrated by metabolic activation in a liquid medium. In other set of experiments, the effect of chemical composition on the expression of mutagenicity in the standard Ames assay was estimated. Premutagens requiring metabolic activation were added to the basic and PNA fractions, and the numbers of revertants obtained in the presence of the fractions were compared with mutation induced by the compounds alone. The basic fraction did not interfere with the mutagenicity of 2-aminoanthracene and 7,9 dimethylbenz[c]acridine. Moreover, in certain experiments, the mutagenicity of the complex fraction plus the added compound was higher than expected on the basis of assays performed on these materials separately. Conversely, the PNA fraction prevented or strongly inhibited mutation by several polynuclear aroumatic compounds, and an acridine. However, the PNA fraction did not inhibit mutation induced by 2-aminoanthracene. The effect of the basic fraction on stability of the S9 enzymes in the standard Ames test was also determined.

Hydrogen formation in nearly stoichiometric amounts from glucose by a Rhodopseudomonas sphaeroides mutant.

Rhodopseudomonas sphaeroides produces molecular H2 and CO2 from reduced organic compounds which serve as electron sources and from light which provides energy in the form of adenosine 5'-triphosphate. This process is mediated by a nitrogenase enzyme. A mutant has been found that, unlike the wild type, will quantitatively convert glucose to H2 and CO2. Techniques for isolating other strains capable of utilizing other unusual electron sources are presented. Metabolism of glucose by the wild-type strain leads to an accumulation of gluconate. The isolated mutant strain does not appear to accumulate gluconate.

Photosystem II regulation of macromolecule synthesis in the blue-green alga Aphanocapsa 6714.

Polymers synthesized by heterotrophically growing (glucose as carbon source) cultures of Aphanocapsa 6714 were compared with polymers synthesized in photosynthetically grown cultures. Loss of photosystem II by dark incubation, or inhibition of light-grown cells with the photosystem II-specific inhibitor dichlorophenylmethylurea, caused an 80 to 90% reduction in the rate of lipid and total ribonucleic acid synthesis, and more than a 90% reduction in the rate of protein synthesis. In contrast, glycogen synthesis was reduced only about 50% in dark cells and less than 30% in dichlorphenylmethylurea-inhibited cells. After longer heterotrophic growth, glycogen became the major component, whereas in photosynthetically grown cultures protein was the major constituent. 14C (from 14CO2 and/or [14C]glucose) assimilated into protein by heterotrophically grown cells was found in amino acids in nearly the same proportions as in photosynthetically grown cells. Thus, routes of biosynthesis available to autotropic cells were also available to heterotrophic cultures, but the supply of carbon precursors to those pathways was greatly reduced. The limited biosynthesis in heterotrophic cells was not due to a limitation for cellular energy. The adenylates were maintained at nearly the same concentrations (and hence the energy charge also) as in photosynthetic cells. The concentration of reduced nicotinamide adenine dinucleotide phosphate was higher in heterotrophic (dark) cells than in photosynthetic cells. From rates of CO2 fixation and/or glycogen biosynthesis it was determined that stationary-phase cells expended approximately 835, 165, and less than 42 nmol of adenosine 5'-triphosphate per mg (dry weight) of algae per 30 min during photosynthetic, photoheterotrophic, and chemoheterotrophic metabolism, respectively. Analysis of the soluble metabolite pools in dark heterotrophic cultures by double-labeling experiments revealed rapid equilibration of 14C through the monophosphate pools, but much slower movement of label into the diphosphate pools of fructose-1,6-diphosphate and sedoheptulose-1,7-diphosphate. Carbon did flow into 3-phosphoglycerate in the dark; however, the initial rate was low and the concentration of this metabolite soon fell to an undetectable level. In photosynthetic cells, 14C quickly equilibrated throughout all the intermediates of the reductive pentose cycle, in particular, into 3-phosphoglycerate. Analysis of glucose-6-phosphate dehydrogenase in cell extracts showed that the enzyme was very sensitive to product inhibition by reduced nicotinamide adenine dinucleotide.

Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714.

Cells of Aphanocapsa 6714 were subjected to alternating ligh-dark periods (flashing-light experiments). The corresponding activation (in the light) and inactivation (in the dark) of the reductive pentose cycle was measured, in vivo, from initial rates of 14CO2 incorporation and also by changes in the total concentration of 14C and 32P in soluble metabolites. Two principle sites of metabolic regulation were detected: (i) CO2 fixation was inactivated 15 to 20 s after removal of the light source, but reactivated rapidly on reentering the light; (ii) hydrolysis of fructose-1,6-diphosphate (FDP) and sedoheptulose-1,7-diphosphate (SDP) by their respective phosphatase(s) (FDP + SDPase) was rapidly inhibited in the dark but only slowly reactivated in the light. The time required for reactivation of FDP + SDPase, in the light, was on the order of 20 to 30 s. As a consequence of the timing of these inactivation-reactivation reactions, newly fixed CO2 accumulated in the FDP and SDP pools during the flashing-light experiments. Changes in the concentrations of the adenylate pools (mainly in the levels of adenosine 5'-triphosphate and adenosine diphosphate) were fast in comparison to the inactivation-reactivation reactions in the reductive pentose cycle. Thus, these regulatory effects may not be under the control of the adenylates in this organism. The activation of CO2 fixation in the light is at least in part due to activation of phosphoribulokinase, which is required for formation of ribulose-1,5-diphoshate, the carboxylation substrate. Phosphoribulokinase activity in crude extracts was found to be dependent on the presence of strong reducing agents such as dithiothreitol, but not significantly dependent on adenylate levels, although adenosine 5'-triphosphate is a substrate.

Efficiency of energy conversion by aerobic glucose metabolism in Aphancapsa 6714.

Efficiency of adenosine triphosphate (ATP) formation from glucose oxidation in Aphanocapsa 6714 was estimated by quantitative measurement of phosphorylated intermediary metabolites and glycogen (polyglucose) formed from (14)C-glucose. P/2e ratios based on oxygen uptake ranged from 2.62 to 3.08, whereas those based on (14)CO(2) evolution ranged from 1.66 to 1.72. The synthesis of glycogen, which is the dominant energy-consuming process in resting cells exposed to exogenous glucose, was almost totally inhibited under anaerobic conditions, and the cellular concentration of ATP decreased steadily. Thus, both net synthesis of ATP and the steady-state concentration of ATP are obligatorily linked to respiration in this heterotrophic unicellular blue-green alga.

Kinetics of glucose incorporation by Aphanocapsa 6714.

Photoautotrophic metabolism of CO(2) was compared with glucose metabolism in the facultative unicellular blue-green alga, Aphanocapsa 6714. Glucose-fed cells incorporated more (14)C into phosphorylated sugar intermediates of the reductive and oxidative pentose phosphate cycles than autotrophic cells. The relative increases were: 140-fold in dark cells; 32-fold in dichlorophenylmethylurea (DCMU)-inhibited cells; and 16-fold in cells assumilating glucose during photosynthetic carbon reduction. On the other hand, incorporation of (14)C from glucose into 3-phosphoglycerate and the amino acid pools of glutamate and aspartate was reduced in dark cells. Rates of protein synthesis in dark and DCMU-inhibited cells were reduced 50 and 80% compared to photoautotrophic cells. In cells assimilating glucose during photosynthesis, rates of (14)C incorporation into the two amino acids and protein were the same as in photoautotrophic cells. Chase experiments, using an excess of (12)C-glucose and CO(2), revealed slow turnover of carbon in dark cells and intermediate turnover rates in DCMU-inhibited cells, when compared to cells assimilating glucose during photosynthesis.

Kinetic properties of phosphotransacetylase from Veillonella alcalescens.

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.

Regulatory properties of acetokinase from Veillonella alcalescens.

Acetokinase from Veillonella alcalescens catalyzes the virtually irreversible synthesis of adenosine triphosphate from acetyl phosphate and adenosine diphosphate. Kinetic analysis revealed that the enzyme was activated by acetyl phosphate and inhibited by adenosine triphosphate. Velocity curves obtained with increasing amounts of adenosine diphosphate were of the Michaelis-Menten type (rectangular hyperbolas) under all conditions employed. However, velocity curves generated by varying the level of acetyl phosphate were sigmoidal, suggesting homotropic interactions for this substrate. Alteration of the pH of the reaction mixture from 7.4 to 10 or addition of a substrate analogue, propionyl phosphate, resulted in the loss of cooperativity and changed the values of the Hill coefficient from 3 to 1. Data obtained in experiments with propionyl phosphate suggested the existence of separate effector sites on the enzyme. The inhibition curves for adenosine triphosphate were also sigmoidal with Hill coefficients ranging between 1 and 3, depending on the concentration of acetyl phosphate. Sedimentation studies with the partially purified enzyme indicated a polymeric structure with a maximum molecular weight of about 90,000 daltons; a subunit of approximately 29,000 daltons was also detected.