Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were essentially free of other proteins as judged by SDS/PAGE. Partial reconstitution of 7-ethoxycoumarin de-ethylase activity was achieved using rabbit NADPH: cytochrome P450 reductase and purified preparations containing P450-B.

The mixed-function oxidases that metabolize dimethylnitrosamine, aminopyrine, benzphetamine, 7-ethoxycoumarin and benzo[alpha]pyrene were measured in adults of the Canton-S, Oregon-R and Hikone-R strains of Drosophila melanogaster. The expression of these activities is both genotype and age dependent.

Electrophoresis of Drosophila microsomes resolves two major heme-containing protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b.

The relationship between dimethylnitrosamine (DMN) demethylase activity and DMN-induced mutagenesis was investigated in Drosophila melanogaster. The activity of DMN-demethylase was at least 10-fold greater in the Hikone-R strain than in three other Drosophila strains. However, the sex-linked recessive lethal (SLRL) mutations induced by DMN in the four strains differed by less than 2-fold. Several possibilities to explain the lack of correlation between DMN-demethylase activity and DMN-induced mutations were tested and eliminated. They include: (i) the presence of inhibitors of DMN-demethylase in extracts of low-activity strains, (ii) a sex bias in the Hikone-R strain in which the enzyme activity is confined to the females, (iii) the possibility that DMN treatment induces DMN-demethylase activity in the low-activity strains and (iv) the possibility that Hikone-R has a much more efficient DNA repair system than the other strains. The results are discussed in terms of what is known about the role of DMN-demethylase in the metabolic activation of DMN in other systems.

Benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-acetylaminofluorene, 2-aminoanthracene, and 1-aminopyrene, when fed to adult Drosophila melanogaster males, gave a negative mutagenic response in the X-linked recessive lethal assay. Benzo[a]pyrene was also ineffective in inducing "Minutes". Aflatoxin B1, EMS and DMN gave a positive response which was dependent on the concentration of mutagen fed. Whole fly homogenates prepared from adult Drosophila were assayed for mixed-function oxidase activity in the Salmonella/microsome test. Crude Drosophila microsomes activated 2-acetylaminofluorene, 2-aminofluorene, 2,7-diaminofluorene, 2-aminoanthracene, 1-aminopyrene, and aflatoxin B1. Tests with benzo[a]pyrene, pyrene, 1,2,3,4-dibenz[a]anthracene, and 7-12-dimethylbenz[a]anthracene were negative.

In a multidisciplinary effort, the authors are attempting to establish a data base for toxicity evaluation of a variety of aqueous effluents and aqueous extracts from solid wastes from fossil fuels, synthetic fuels, and shale oil derived fuels. In genetic toxicology testing, short-term mutagenicity tests, including bacterial, fungal, insect, and mammalian cell systems, have been applied in a comparative sense to exemplary test materials. The Salmonella histidine reversion assay (Ames test) has been shown to be generally applicable, especially when utilized with chemically fractionated materials. Liquid-liquid extraction and column chromatography are used to separate crude test materials into defined fractions for bioassay, paralleled by chemical analyses. The test materials have included various crude oils and product waters along with extracts from raw shale and processed shale. The mutagenic materials have been observed and quantitated. Extrapolations to specific compounds and to the overall biological hazard of the test materials are in progress. Comparative studies with samples from existing petroleum technologies and fossil fuel processes are being carried out.

The mutagenic activity of 7 nitrosopiperazines, 2 nitropyrrolidines, and 3 nitrosomorpholines was examined in the X-linked recessive-lethal assay of Drosophila melanogaster. Mutagenicity is also reported for a series of cyclic nitrosamines that differ in structure only in the number of carbon atoms in the ring. Of the 18 compounds tested, 6 (nitrosopiperazine; 2,3,5,6-tetramethyldinitrosopiperazine; nitrosoproline; 2,5-dimethylnitrosopyrrolidine; nitrosothiomorpholine; and nitrosooctamethyleneimine) were nonmutagenic. As we reported earlier in investigations with the nitrosopiperidines, substitutions with methyl groups at all of the alpha-carbon atoms reduce or eliminate the mutagenic activity of dinitrosopiperazine and nitrosopyrrolidine.

N-Nitrosopiperidine (NP) and various derivatives were fed to Drosophila melanogaster males over a wide concentration range in order to assess their mutagenic potency in the induction of X-linked recessive lethals and chromosome loss. NP was effective in inducing lethals, as were its halogen and methylsubstituted derivatives, with the exception of 2,6-dimethyl NP. (Methyl substitutions at the alpha carbon atoms reduce or eliminate mutagenic activity.) Substitutions of halogen groups on the piperidine ring enhanced the mutagenic activity, with the 3-chloro compound being the most mutagenic. In contrast, substitutions with a hydroxyl, carboxyl, or keto group resulted in a loss of mutagenicity. None of the compounds tested increased the frequency of chromosome loss or breakage in mature sperm.

As research expands the types of energy sources for the future, there is a need to understand the health impacts of fuels and their emissions and to understand what health-research data gaps exist so that in the future proper and informative research and decision-making can be done. In that regard, this series of papers will explore what is known about the history, carcinogenicity, and genotoxicity of fuels and their emission products and attempt to identify major data gaps and areas of interest for future research. The reviews will concentrate on petroleum-derived fuels and biofuels. Although the length of these papers may cause the reader to think otherwise, the coverage of published works is intended to be illustrative rather than exhaustive and is intended for a multidisciplinary audience. This series of papers is not a risk assessment; instead, it is an attempt to introduce the reader with the history and terminology needed when examining fuels and emissions for genotoxic effects. The purpose of this particular paper is to provide a background for the other papers (both within this series and within papers by others) and to establish some principles used in these reviews. In particular, this paper provides definitions, general histories relevant to the topic, an overview of the regulatory history, and appendices the author believes are useful to those interested in the fields associated with the toxicology of carbonaceous fuels and their emissions.

In an effort to gather information on the potential genetic hazards of existing or proposed energy-generating or -conversion systems, we have begun a correlated analytical and genetic analysis of a number of technologies. The work is divided into two phases: one deals with known compounds expected to occur in the environment through energy production, conversion, or use; the other deals with actual samples from existing or experimental processes. To approach the problems of coping with and testing large numbers of compounds, we set up a form of the "tier system." Operating units utilizing Salmonella, Escherichia coli, yeast, human leukocytes, mammalian cells, and Drosophila have been initiated. Various liquid-liquid extraction methods and column chromatographic separations have been applied to crude products and effluents from oil-shale, coal-liquefaction, and coal-gasification processes. Mutagenicity of the various fractions is assayed by means of reversion of histidine-requiring auxotrophs of Salmonella typhimurium; comparative studies are carried out with the other genetic systems. In order to incorporate metabolic activation of these fractions and compounds, rat liver homogenates (S-9) are used in the various assays. Results implicate chemicals occurring in the basic (ether-soluble) and the neutral fractions as potential genetic hazards. Chemical constituents of these fractions (identified or predicted) were tested individually for their mutagenic activity.

Hindered amines such as 2,2,6,6-tetramethyl piperidines and N-methyl-2,2,6,6-tetramethyl piperidines and hindered amino ethers such as N-methoxy-2,2,6,6-tetramethyl piperidines were examined with respect to their reactivity towards triplet-excited benzophenone (3BP∗) in Ar-saturated acetonitrile solution at room temperature. Upon measuring phosphorescence decay rates in the absence and presence of quencher it was found that the amines are two to three orders of magnitude more reactive than the amino ethers. For all amine/BP systems examined the generation of free ions was inferred from an increase in the electrical conductivity. This indicates that a charge transfer (CT) mechanism is operative in these cases. Notably, tertiary amines were found to be more reactive towards 3BP∗ and give higher free ion yields than secondary amines. In the case of amino ether/BP systems evidence for a CT mechanism was not obtained in spite of an increase in the electrical conductivity indicating the generation of free ions. The latter are formed much faster than the phosphorescence decays and very likely originate from triplet–triplet annihilation (3BP∗+3BP∗→BP++BP−).