T.K. Rao

Polycyclic aromatic primary amines as determinant chemical mutagens in petroleum substitutes

Petroleum crude oils and coal- and shale-derived petroleum substitutes have been separated by chemical class and the class fractions have been subjected to bacterial mutagenicity testing. Alkaline constituents of the petroleum substitutes are found to make major contributions to their mutagenicities. High-resolution chromatographic and spectroscopic analysis of alkaline subfractions enriched in mutagenic activity show the causative agents to be polycyclic aromatic primary amines. The amines may be responsible for the increased biological activity of coal- and shale-derived petroleum substitutes relative to petroleum.

Mutagenicity of N-nitrosopiperidines with Salmonella typhimurium/microsomal activation system

Abstract Using Salmonella typhimurium tester strains, we have examined N -nitropiperidine and various substituted nitrosopiperidines for their mutagenic potency. Most of the nitrosopiperidines require metabolic activation. Phenobarbital appears to be the most effective inducer of the rat liver enzymes. We also observed a correlation between mutagenicity and carcinogenic potency of these compounds. The carbon atoms alpha to the N -nitroso group seem important since blockage of those positions reduces or eliminates mutagenicity as well as carcinogenicity of the nitrosopiperidine.

Mutagenicity of 4,4′-MethyIenedianiline Derivatives in the Salmonella histidine reversion assay

Abstract4,4′-Methylenedianiline and its derivatives were assayed for mutagenicity in the Salmonella/microsomal mutagenicity assay developed by Ames. A specificity to revert strain TA98 suggests a mechanism of frameshift mutagenesis. Liver microsomal preparations (S-9) from rats induced with phenobarbital were most effective for metabolic activation. Alkyl substitution of 4,4′-methylenedianiline did not alter its mutagenic activity; however, substitution of both positions ortho to the amino group eliminated mutagenic activity. Substitution with alkoxy-carbonyl groups eliminated mutagenic activity, whereas halogen substitution (chlorine, fluorine) enhanced the mutagenic activity. The results presented here show the use of structure-activity studies as predictive tools for the assessment of genotoxic properties of industrial chemicals.

Comparative mutagenesis of quinolines

Abstract Quinoline, 8-hydroxyquinoline, 8-nitroquinoline, and 8-aminoquinoline are mutagenic in Salmonella when assayed by histidine reversion. Metabolic activation is required for the maximal effect. Frameshift mutagenesis appears to be the mechanism with 8-nitro- and 8-aminoquinoline. Quinoline and 8-hydroxyquinoline affect only the sensitive TA100 (missense, R factor) strain. 8-Amino-and 8-hydroxyquinolines induce chromatid aberrations in human leukocytes. 8-Nitroquinoline has no effect at the concentrations tested. 8-Aminoquinoline is not effective in yeast when assayed by forward mutation, gene conversion, or mitotic recombination; fluctuation tests show a weak effect. 8-aminoquinoline is not an effective mutagen in Drosophila when assayed for sex-linked recessive lethals by an adult feeding method.

Separation of neutral nitrogen compounds from synthetic crude oils for biological testing

Isolation of neutral N-PAHs (PAH homologues containing one or more ring nitrogens) is achieved in three steps using acid-base extraction, gel filtration on Sephadex LH-20, and adsorption chromatography on silicic acid. Gas chromatographic/mass spectrometric analysis of the neutral N-PAH fractions indicated the following as major components: C1-C3 phenylpyrroles, indole, C1-C6 indoles, C1-C3 phenylindoles, carbazole, C1-C5 carbazoles, benzocarbazoles, and C1-C3 benzocarbazoles. The neutral N-PAH fractions were subjected to mutagenicity tests using Salmonella typhimurium/microsomal activation systems devised by Ames. The neutral N-PAH fraction of the coal-derived oil had a specific activity more than twice that of the PAH fraction of the same oil, whereas the shale oil neutral N-PAH fraction showed no activity. These results are discussed in the context of previous work with these oils and with some pure neutral N-PAH compounds.

Separation and Identification of Mutagenic Constituents of Petroleum Substitutes

Abstract A study combining chemical separations, mutagenicity testing, and spectroscopic identifications is underway to isolate and identify mutagens in coal-and shale-derived oils. Ether-aqueous partition combined with Sephadex LH-20 chromatography of the resulting neutral fraction is introduced as a preferred class fractionation procedure. The uniquely important role of polycyclic aromatic primary amines in the mutagenicity of petroleum substitutes is reviewed. Questions are raised concerning the role of polycyclic aromatic hydrocarbons in the mutagenicity of the neutral fraction of petroleum substitutes.

Characterization of Mutagenic Coal Fly Ash and Extracts

Post-electrostatic precipitator (ESP) fly ash samples were collected from a coal-fired electric power generation plant under three modes of plant operation: normal operation, a low NOx-emission mode of combustion, and operation with the ESP shorted-out. Results of chemical and physical characterization of the ashes were compared with bacterial mutagenicity bioassay to determine parameters or compounds correlating with bioactivity. The general physical properties, ultimate composition, and trace elemental and radiochemical species determined did not correlate with the mutagenicity. Only the presence of aromatic hydrocarbons and chemically derivatizable polar organic compounds appeared to be associated with mutagenicity of the fly ash.

Mutagenicity of N-nitrosopyrrolidine derivatives in Salmonella (Ames) and Escherichia coli K-12 (343/113) assays.

The mutagenicity of nitrosopyrrolidine (NPYR) and its derivatives was determined by use of the Ames Salmonella assay. A clear specificity to revert the missense stain of TA1535 and a requirement for the phenobarbital-induced rat-liver activation system (S9 mix) were noted. 3,4-Dichloronitrosopyrrolidine was more mutagenic than NPYR, whereas 3-hydroxynitrosopyrrolidine was weakly mutagenic. The carcinogenic nitroso-3-pyrrolidine was not mutagenic under the test conditions. The noncarcinogenic derivatives (2,5-dimethylnitrosopyrrolidine, nitrosoproline and 4-hydroxynitrosoproline) were not mutagenic. Liquid preincubation assays were not any more effective than the pour-plate assays. Selected derivatives of NPYR were tested in the Escherichia coli K-12 (343/113) assay A specificity to revert the missense mutation at the arg locus and a dependence on phenobarbital-induced rat-liver S9 mix were noted with NPYR and its derivatives. 3,4-Dibromonitrosopyrrolidine, which was not mutagenic in Salmonella, was effective in E. coli, and the weakly carcinogenic NPRL was a weak mutagen resulting in a 2-fold enhancement in the E. coli arginine reversion assay.

Short-Term Bioassay of Complex Organic Mixtures: Part II, Mutagenicity Testing

The feasibility of using short-term mutagenicity assays to predict the potential biohazard of various crude and complex test materials has been examined in a coupled chemical and biological approach. The principal focus-of the research has involved the preliminary chemical characterization and preparation for bioassay, followed by testing in the Salmonella histidine reversion assay described by Ames (1). The mutagenicity tests are intended to (a) act as predictors of profound long-range health effects such as mutagenesis and/ or carcinogenesis, (b) act as a mechanism to rapidly isolate and identify a hazardous biological agent in a complex mixture, and (c) function as a measure of biological activity correlating baseline data with changes in process conditions. Since complex mixtures can be fractionated and approached in these short-term assays, information reflecting on the actual compounds responsible for the biological effect may be accumulated. Thus, mutagenicity tests will (d) aid in identifying the specific hazardous compounds involved and in establishing priorities for further valid testing, testing in whole animals, and more definitive chemical analysis and monitoring.

Preparation of oils for bacterial mutagenicity testing

4 procedures used to prepare fossil-derived oils for bacterial mutagenicity testing have been examined. These are, (a) dewaxing by partitioning the oil between dimethyl sulfoxide (DMSO) and cyclohexane, (b) incorporating a surfactant to increase compatibility of the oil with the bioassay media, (c) directly slurrying the oil in DMSO, and (d) computing the mutagenicity of the oil by summing the contributions of individual chemical class fractions. DMSO slurries generally exhibit higher mutagenicities than computed by summing the contributions of chemical class fractions. Results of testing DMSO-slurries correlate (r = 0.87) well, however, with those obtained by summation. Mutagenicity results agree within a factor of two for the samples tested by 4 sample preparation procedures.