David A. Haugen

Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation

We observed that complex mixtures of aromatic compounds isolated from a coal-derived oil suppressed the mutagenic activity of the indirect mutagens benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-aminofluorene, and 2-acetylaminofluorene as measured in the Salmonella/microsome mutagenicity assay, using strain TA98 and metabolic activation with Aroclor-induced rat-liver S9 or microsomes. The mixture also inhibited S9-dependent benzo[a]pyrene metabolism and covalent binding to DNA in a cell-free system. The mixture did not suppress the activity of either the direct acting mutagens 2-nitrofluorene and benzo[a]pyrene diol-epoxide, or of the indirect mutagen N-hydroxy-2-acetylaminofluorene which requires a microsomal deacetylase for metabolic activation. Spectrophotometric measurements showed that components of the mixture bound to microsomal cytochrome P-450. The mixture did not inhibit microsomal NADPH-cytochrome c (P-450) reductase. These observations show that the mixtures inhibited metabolic activation by the microsomal monooxygenase system, probably by binding of unidentified components to cytochrome P-450. The resulting inhibition of mutagenesis may have implications for risk estimates for the mixtures we examined as well as for other types of complex mixtures for which similar inhibitory effects have been observed.

Toxicologic Responses to a Complex Coal Conversion By-Product: Mammalian Cell Mutagenicity and Dermal Carcinogenicity

The overall toxicity of complex mixtures cannot be predicted simply by adding the toxicities of known toxic compounds in the mixture. The toxicity of some of the components in the mixture is often unknown and synergisms and antagonisms, which cannot be predicted solely on the basis of the chemical composition, may occur. To better understand the chemical and biochemical interactions that modify the ultimate toxicity of mixtures, we are isolating and characterizing well-defined chemical classes of chemicals and determining the toxicity of these chemical class fractions with in vivo andin vitro bioassays. The need for this approach with coal conversion materials has recently been demonstrated by interactive effects observed in both in vitro and in vivo toxicity assays. For example, a mixture of polycyclic aromatic hydrocarbons from coal conversion materials inhibited the bacterial mutagencity of benzo[a]pyrene (Haugen and Peak, 1983) and potentiated the mutagenicity of 2-aminoanthracene (Pelroy and Peterson, 1979; Kawalek and Andrews, 1981) as measured in the Salmonella/microsome assay. In addition, tumorigenic responses to carcinogenic components of coal conversion materials were greater than those determined for the whole material (Mahlum, 1984).

Promotion of preneoplastic changes in liver by coal-derived organic mixtures applied to skin

The promotion of preneoplastic hepatocyte foci was observed in rats neonatally initiated by a single intraperitoneal injection of benzo[a]pyrene (BP) or diethylnitrosamine (DEN) and exposed, from weaning, to repeated topical applications of coal-derived complex organic mixtures that are carcinogenic for mouse skin. Topical application of these mixtures in the absence of prior initiation did not cause significant induction of hepatocyte foci. These observations indicate the advantage of the neonatal rat hepatocarcinogenesis system for detecting promoting activity in carcinogenic mixtures and identify the existence of systemic tumorigenic risk from cutaneous contact with promoting agents.

Phenotypically selective promotion of diethylnitrosamine-initiated altered hepatocyte foci by dietary phenobarbital or a topically applied coal-derived organic mixture in male and female rats☆

Relative frequencies of diethylnitrosamine (DEN)-initiated foci of altered hepatocytes appearing in response to promotion by either dietary phenobarbital or a topically applied coal-derived organic mixture (CDM) were investigated in male and female rats. The focus population was examined for two histochemical markers, elevated gamma-glutamyl transpeptidase [GG(+)] and iron exclusion [FE(-)], giving rise to 3 detectable focus phenotypes, i.e., GG(+) foci, FE(-) foci, and GG(+)/(FE(-) foci. Frequencies of the 3 phenotypes were quantitated through the use of serial frozen sectioning and computer-assisted image analysis. In agreement with our prior observations, cutaneous exposure to CDM or dietary phenobarbital promoted the expression of DEN-initiated foci. However, the current data showed that this promoting effect of CDM occurred only in females and was restricted to foci with the GG(+)/FE(-) phenotype. Dietary phenobarbital, on the other hand, promoted both the GG(+) and GG(+)/FE(-) phenotypes and was effective in both males and females, although a sex-related differential in the promoting efficiency of phenobarbital was also observed. The pronounced heterogeneity in the responses of the 3 focus phenotypes suggests that each phenotype is the consequence of a specific type of genomic alteration with a specific capacity to undergo phenotypic expression in response to a given promoting stimulus.

Protection by uridine diphosphoglucuronic acid and DT-diaphorase against the cytotoxicity of polycyclic aromatic hydrocarbons isolated from a complex coal gasification condensate

The cytotoxicities of polycyclic aromatic hydrocarbon (PAH) subclasses isolated from a complex organic mixture (coal gasification condensate) were studied in vitro in Chinese hamster ovary cells, in the presence of rat liver microsomes from animals pretreated with Aroclor. Toxicity was enhanced by microsomal metabolism and was inversely related to aromatic ring number. Rat liver cytosol, semipurified DT-diaphorase, and uridine diphosphoglucuronic acid decreased the cytotoxicity of a variety of PAH mixtures and representative PAH, as well as individual PAH metabolites. The results indicate that the in vitro toxicity of complex PAH mixtures is caused primarily by hydroxy-PAH and quinone metabolites of the predominant, nonmutagenic two- and three-ring PAHs.

Isolation of a highly mutagenic aminophenanthrene from a coal gasification process tar.

A major portion of the mutagenic activity associated with products and by-products of coal conversion can be ascribed to nitrogen-containing bases. We improved the extraction efficiencies for three- to five-ring aromatic bases by extracting them with a mixture of methanol and aqueous HCl, rather than with aqueous HCl alone. A complex mutagenic basic fraction of a coal gasification process tar was successively fractionated using cation exchange and reversed phase high-performance liquid chromatography. The fractions were assayed for mutagenic activity and were chemically analyzed by gas chromatography and gas chromatography-mass spectrometry. Aminophenanthrenes were identified as major contributors to the mutagenicity of the basic fraction. Aminonaphthalenes, aminobiphenyls, and their alkyl homologs were also present but were not detected as principal mutagens.

Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines Non-specific reaction with phenols and benzo[a]pyrene ☆

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.

Formation of hemoglobin-benzo[a]pyrene adducts in human erythrocytes incubated with benzo[a]pyrene and hamster embryo cells

Evidence is accumulating that the levels of covalent carcinogen-macromolecule adducts, including adducts with hemoglobin, reflect biologically effective levels of carcinogen exposure. The purposes of the present study were (a) to establish a cellular system for obtaining adducts between intracellular human hemoglobin and metabolites of polycyclic aromatic hydrocarbons (PAH), and (b) to evaluate techniques for chromatographic characterization of the adducts. We showed that hemoglobin-benzo[a]pyrene adducts were formed when human erythrocytes were treated with [3H]benzo[a]pyrene (BP) in the presence of hamster embryo fibroblasts, which are known to be effective for BP metabolism. After lysis of the erythrocytes, noncovalently bound BP and its metabolites were effectively removed from hemoglobin under mild conditions by using hydrophobic interaction and size-exclusion liquid chromatography. Three to five distinct adducts were resolved by reversed-phase and ion-exchange liquid chromatography. As determined by a two-step, reversed-phase liquid chromatographic procedure, trypsin treatment of globin from the cellular system yielded at least three of the four 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy BP tetrols known to arise from mammalian metabolism of BP. This observation is consistent with both (a) the recently described formation of labile carboxyl esters via reaction of BP-7,8-dihydrodiol-9,10-epoxide (BPDE) with hemoglobin and (b) the known formation of both anti- and syn-BPDE in hamster embryo fibroblasts. In addition, high-performance liquid chromatographic analysis demonstrated the presence of other products presumed to be BP-peptide adducts because of their susceptibility to thermolysin treatment.

Photochemistry of 4-thiouridine and thymine

Abstract— When thymine is irradiated in aqueous solution with monochromatic 334‐nm UV radiation in the presence of 4‐thiouridine a photoproduct of thymine is formed, as shown by thin‐layer chromatography and autoradiography. The quantum yield for the formation of thymine photoproduct (θ=0.017) is greater than that for cytosine photoproduct formation (θ= 0.0015). The identity of the photoproduct is not known: one possibility is the formation of an adduct between the sensitizer and the base yielding a pyrimidine‐pyrimidone type of photoproduct.

Isolation and identification of mutagenic polycyclic aromatic hydrocarbons from a coal gasifier condensate

An iterative scheme of short-term bacterial mutagenesis assays and multidimensional liquid chromatographic methods was used to isolate mutagens from a neutral fraction of a complex carcinogenic tar formed as a by-product of coal gasification. Mutagens in the complementary reversed- and normal-phase liquid chromatographic fractions were identified by capillary column gas chromatography and gas chromatography-mass spectrometry. The principal mutagens included a variety of 4- to 6-ring unsubstituted polycyclic aromatic hydrocarbons, their alkylated homologs, and related methylene-bridged compounds. Most of the unsubstituted mutagens were known carcinogens that were qualitatively similar to those detected by carcinogenicity assays of fractions from other types of coal tars and cigarette smoke condensate. The chromatographic approach is generally applicable to complex mixtures and is especially well suited for fractionating mixtures of aromatic compounds and their alkyl homologs.