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Structural modifications of cellular macromolecules by chemical carcinogens may represent early and requisite events in neoplastic transformation (1, 2). Through interactions of this nature, qualitative changes could be induced in informational macromolecules such as DNA and RNA, and these lesions could provide a molecular basis for alteration of gene expression in carcinogenesis. Identification of the products of these reactions (herein referred to as adducts) is essential in order to: (i) gain insights into mechanisms of carcinogen activation; (ii) determine the reactive centers in these macromolecules; (iii) follow the kinetics of appearance and disappearance of adducts in the cell; and (iv) relate specific patterns of macromolecule modification with the ultimate development of tumors in target organs of susceptible species.Aflatoxin B1 (AFB1) is a very potent liver carcinogen in several animal species (3), and epidemiologic evidence indicates that it is also an important factor in the etiology of human liver cancer in certain sections of the world (4). AFB1 binds covalently to cellular macromolecules, including DNA, in mvo (5-7) and in vitro after metabolic activation (8-10). The relationship of this type of interaction to its mechanism of action has been emphasized (11). Strong indirect evidence has indicated the production of AFB1-2,3-oxide as a major activated metabolite responsible for macromolecular binding in vitro and in vivo (5-7, 9, 12), but structures of specific adducts formed with nucleic acids or proteins have not been determined. The purpose of the research reported here was to determine the structure of the major adduct formed with DNA by AFB1 activated metabolically in vitro. The results indicate that approximately 90% of the binding in vitro can be attributed to a single adduct, which was isolated in sufficient quantity for structural analysis and identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (structure I).,H0 Ho (c) H3C (c) Hk I MATERIALS AND METHODS Liver microsomes used for metabolic activation of AFB1 were prepared from phenobarbital-treated male Fischer rats (13) by the procedure of Kinoshita et al. (14). The incubation mixture (400 ml) for the binding of AFB1 to DNA included Tris-HCl (pH 7.5,45 mM), MgCl2 (3 mM), glucose-6-phosphate (5 mM), NADP (0.8 mM, Sigma Chemical Co.), glucose-6-phosphate dehydrogenase (0.4 unit/ml, Sigma Chemical Co.), approximately 1 mg of microsomal protein per ml, calf thymus DNA (20 A260 units/ml or a total of 340 mg; type I, Sigma Chemical Co.), AFB1 [224 ,uM added Abbreviations: AFB1, aflatoxin B1; I, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin Bj; II, 2,3-dihydro-3-hydroxy-2-(4-nitrobenzoxy)-aflatoxin B1; HPLC, high-pressure liquid chromatography; NMR, nuclear magnetic resonance; FD, field-desorption mass spectrometry; EI, electron-impact mass spectrometry.

While it is known that urban airborne particles typically contain trace levels of bacterial mutagens and rodent carcinogens, little work has been done to identify chemicals in such particles that can genetically alter human cells. In this paper, we describe the analysis of an organic extract of a Washington, DC, airborne particle sample (SRM 1649) for human cell mutagens. Due to the chemical complexity of the extract, a bioassay-directed fractionation method was used to separate mutagenic constituents into chemically simplified fractions. Mutagenicity testing was done using the h1A1v2 cell line, a line of human B-lymphoblastoid cells that have been engineered to overexpress the human cytochrome P4501A1. Chemical analysis of mutagenic fractions was accomplished using GC−MS and HPLC−UV techniques. Our results indicate that ∼20% of the total mutagenicity the extract was accounted for in two fourth-order fractions that contained ∼3% of the total extract mass. These fractions were composed largely of polycyclic aromatic hydrocarbons (PAH). A total of 13 PAH were identified that accounted for ∼15% of the mutagenicity of the extract. Of these, the most important mutagens were cyclopenta[cd]pyrene, benzo[a]pyrene, and benzo[b]fluoranthene, accounting for ∼7, ∼4, and ∼2%, respectively, of the extract mutagenicity. Naphtho[2,1-a]pyrene (N[2,1-a]P) and naphtho[2,3-a]pyrene (N[2,3-a]P), two previously unknown potent human lymphoblast mutagens, were also identified in the sample. N[2,1-a]P accounted for ∼3% of the extract mutagenicity; N[2,3-a]P, which was present at relatively low levels, accounted for <1% of the extract mutagenicity. The remainder of the mutagenicity was found in fractions that contained more polar compounds. One of these polar fractions contained many different classes of oxygenated polycyclic aromatic compounds (oxy-PAH) including ketones, quinones, coumarins, and carboxylic acid anhydrides; however, of the mutagenic oxy-PAH identified, only the ketone 6H-benzo[cd]pyren-6-one (∼0.5%) was found to account for a significant portion of the total mutagenicity of the extract. Nitro-PAH, many of which are potent bacterial mutagens, did not contribute significantly to the mutagenicity of this sample because they were present at low concentrations and because they are not particularly mutagenic in h1A1v2 cells.

The human cell mutagenicity of Los Angeles airborne fine particulate matter is examined via bioassay-directed chemical analysis. A 1993 composite fine particle sample is separated via liquid chromatography into fractions containing organic compounds of varying polarity. Samples are analyzed by the h1A1v2 human cell mutagenicity assay to identify those fractions that contain human cell mutagens and by GC/MS to identify the chemical character of those mutagens. Those subfractions that contain unsubstituted polycyclic aromatic compounds (PAC) are responsible for a considerable portion of the mutagenic potency of the whole atmospheric sample. Six unsubstituted PAC (cyclopenta[cd]pyrene, benzo[a]pyrene, benzo[ghi]perylene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, and benzo[k]fluoranthene) account for most of the mutagenic potency that can be assigned to specific compounds within the atmospheric samples. Important semipolar mutagens that are quantified include 2-nitrofluoranthene and 6Hbenzo[cd]pyren-6-one. A large number of other aromatic organics are identified as candidates for future testing as pure compounds in the human cell assay, at which time it should be possible to account for more of the mutagenic potency of the atmospheric samples.