Covalent binding of 4,4'-methylenebis-(2-chloroaniline) to rat liver DNA in vivo and of its N-hydroxylated derivative to DNA in vitro.

The binding of [ring-3H]4,4'-methylenebis(2-chloroaniline) (MOCA) to rat liver DNA following i.p. injection is demonstrated. Three discrete adducts were eluted on HPLC following enzymic hydrolysis to the nucleoside level. Three adducts, with the same retention times on HPLC, were present after i.p. injection of the N-acetyl derivative of MOCA tritiated in the benzene rings. Only two of these adducts were found when the N-acetyl derivative, tritiated on the acetyl group, was used. Thus, at least one of the adducts formed by MOCA is not acetylated. The N-hydroxy derivative of MOCA was synthesised and reacted with DNA in vitro. Following enzymic hydrolysis of this DNA, the major product was shown to co-elute with the radiolabelled non-acetylated adduct produced in the liver DNA of animals injected with [ring-3H]MOCA. This same compound was also isolated following the reaction of N-hydroxy-4-amino-3-chlorobenzyl alcohol with DNA, and subsequent enzymic hydrolysis. The NMR and mass spectra of the synthetic adduct were consistent with N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol. Thus, the major adduct formed in vivo has involved cleavage of the bond between the methylene bridge and one of the aromatic nuclei of MOCA.