Effect of Substitution Site upon the Oxidation Potentials of Alkylanilines, the Mutagenicities of N-Hydroxyalkylanilines, and the Conformations of Alkylaniline-DNA Adducts M. Matilde Marques,* ,† Luı ´sa L. G. Mourato, † M. Teresa Amorim, † M. Ame ´lia Santos, † William B. Melchior, Jr., ‡ and Frederick A. Beland* ,‡ Centro de Quı ´mica Estrutural, Complexo I, Instituto Superior Te ´ cnico, Av. Rovisco Pais, 1096 Lisboa Codex, Portugal, and Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received June 17, 1997 X Carcinogenic arylamines typically undergo metabolic activation via N-hydroxylation followed in most instances by O-esterification. In this study, the ability of methyl-, dimethyl-, and ethylaniline constituents of tobacco smoke to undergo oxidation at the nitrogen atom was analyzed. In addition, the mutagenicity of the corresponding N-hydroxyalkylanilines and the conformational properties of the DNA adducts generated from their N-acyloxy derivatives were investigated. All the arylamines underwent irreversible electrochemical N-oxidation at potentials higher than those observed for the oxidation of carcinogenic polynuclear aromatic amines. There were minor differences in the oxidation potentials, which were consistent with the position and electron-donating abilities of the alkyl substituents; however, the differences appeared to be too small to account for the range of genotoxic effects among the alkylanilines. N-Hydroxyarylamines containing p-alkyl substituents had increased mutagenicities in Sal- monella typhimurium TA100, which was attributed to their higher efficiencies of adduct formation. Increased mutagenicities were also observed upon o-alkyl substitution; however, this property was not related to a greater ability of the ortho-substituted species to form DNA adducts, which suggested that adducts from ortho-substituted alkylanilines may be intrinsically more mutagenic than their meta- and para-substituted analogues. In all instances, N-(acyloxy)- arylamines generated from the N-hydroxyarylamines reacted with dG, dG nucleotides, and DNA to yield C8-substituted dG derivatives as the major adducts. The alkylaniline-dG adducts displayed distinct conformational trends that were determined by the location of the alkyl substituents. Spectroscopic data indicated higher percentages of low-energy syn conformers in the adducts that contained alkyl groups ortho to the arylamine nitrogen as opposed to adducts not bearing ortho substituents. The data strongly suggest that the conformational properties of the DNA adducts, in particular their ability to adopt syn conformations, may be determinant factors for the genotoxic responses elicited by certain alkylanilines (e.g., 2-methylaniline and 2,6-dimethylaniline). Introduction Aromatic amines and amides are chemical carcinogens to which human exposure from diverse origins is well- documented (1-4). Cigarette smoke is a significant source of primary aromatic amines, including the human urinary bladder carcinogens 2-naphthylamine and 4-ami- nobiphenyl. Since no other class of compounds identified in cigarette smoke has been implicated in human bladder carcinogenesis (5), aromatic amines are thought to be responsible for the causal relationship observed between cigarette consumption and the onset of bladder cancer (5-7). Aromatic amines have also been suggested to play a role in the induction of breast cancer in postmenopausal women who smoke (8). The major contributors to the arylamine fraction of cigarette smoke are single-ring arylamines such as aniline and its methyl, dimethyl, and ethyl derivatives (2, 3). These compounds have recently been detected in human milk from women smokers (9). In contrast to their binuclear analogues, mononuclear aromatic amines are generally considered to be weak carcinogens (10- 12); however, the risks of chronic exposure to some aniline derivatives may be underestimated. 2-Methyl- aniline (o-toluidine), for example, has long been recog- nized as a rodent carcinogen (13) and may account for the increased risk of bladder cancer observed in rubber industry workers (14). 2,6-Dimethylaniline (2,6-xyli- dine), which exists in cigarette smoke and also as a major metabolite of the potent anesthetic and antiarrhythmic drug lidocaine (15), is carcinogenic in rats (16). The potential carcinogenicity of this arylamine to humans remains uncertain (17). Aromatic amine carcinogens are known to exert their biological effects upon metabolic activation to reactive electrophilic intermediates that bind to DNA, yielding N-(deoxyguanosin-8-yl)arylamines (dG-C8-Ar) 1 as major persistent adducts (18-20). In most instances, the initial step in the activation involves cytochrome P450-mediated N-hydroxylation to N-hydroxyarylamines, which may be further activated via enzymatic O-esterification. Previ- ous studies (21-25) have shown that synthetic analogues † Instituto Superior Te ´cnico. ‡ National Center for Toxicological Research. X Abstract published in Advance ACS Abstracts, November 1, 1997. 1266 Chem. Res. Toxicol. 1997, 10, 1266-1274 S0893-228x(97)00104-5 CCC: $14.00 © 1997 American Chemical Society