Bioactivation of N-hydroxy-2-acetylaminofluorenes by N,O-acyltransferase: substituent effects on covalent binding to DNA.

N-Acetoxyarylamines are reactive metabolites that lead to arylamine adduct formation with biological macromolecules. A series of 7-substituted-N-hydroxy-2-acetylaminofluorenes were converted to reactive N-acetoxyarylamines by enzymatic N,O-acyltransfer in the presence of DNA. The N-arylhydroxamic acid substrates that contained electronegative 7-substituents formed greater amounts of DNA adducts than either the unsubstituted compound (N-OH-AAF) or those analogs that contained electron-donating groups in the 7-position. Glutathione did not decrease the rates of DNA adduct formation, but other nucleophiles, such as potassium O-ethylxanthate, thiourea and N-acetylmethionine, inhibited adduct formation by the 7-Br-substituted compound (7-Br-N-OH-AAF) and the unsubstituted parent compound (N-OH-AAF). Nucleophiles, reducing agents (e.g. ascorbic acid) and spin-trapping agents had minimal effect on DNA adduct formation by the bioactivated form of 7-acetyl-2-(N-hydroxy-acetylamino)fluorene (7-Ac-N-OH-AAF). Triethylphosphite, an agent that reacts with aryl nitrenes, caused a concentration-dependent reduction in the amount of DNA adduct formed subsequent to bioactivation of 7-Ac-N-OH-AAF, but did not influence adduct formation when N-OH-AAF and 7-Br-N-OH-AAF were the substrates. The results indicate that a change in the reaction mechanism(s) responsible for DNA adduct formation occurred when the strongly electronegative acetyl group was incorporated into the 7-position of N-OH-AAF. It is proposed that a nitrene intermediate is involved in the formation of covalent adducts with DNA when 7-Ac-N-OH-AAF is activated by N,O-acyltransfer.