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.

Aflatoxin B1 (AFB1) was shown to react primarily with one or more lysine residues in serum albumin (SA), accounting for more than half of the total binding to this protein. The radioactivity associated with SA following administration of [U-14C]AFB1 to rats was cleared with a half-life of 2.5 days, which is not significantly different from the half-life of unmodified albumin in the normal rat. The product isolated from a Pronase digest of in vivo-modified SA was identical by chromatographic retention time and u.v. and mass spectroscopy to the synthetic product obtained by the acylase-catalyzed deacetylation of the reaction product of N alpha-acetyl-L-lysine with 8,9-dihydro-8,9-dibromo-AFB1. The latter was characterized by u.v., fluorescence, 500 MHz 1H-n.m.r. and fast atom bombardment mass spectrometry. The spectral data strongly support a structure in which the terminal dihydrofuran ring of AFB1 has been converted to a pyrrolinone ring. It is proposed that the initial adduct is formed by condensation of the dialdehyde tautomer of 8,9-dihydro-8,9-dihydroxy-AFB1, with the epsilon-amino group of lysine, to form a Schiff base, and that the Schiff base undergoes an Amadori rearrangement to an alpha-amino ketone. The pyrrolinone ring is formed by condensation of the amino group with the remaining aldehyde to yield the final product. The purified product was relatively stable but was shown to decompose significantly under the conditions used to isolate it from modified SA.

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