Prostaglandin H synthase in the presence of arachidonic acid catalyzes the peroxidative metabolism of 2-aminofluorene (2-AF) to an electrophile(s) which binds covalently to calf thymus DNA in vitro. Moreover, this electrophile(s) appears distinct from the classical 2-AF-derived electrophiles, N-hydroxy-2-AF and the 2-AF nitrenium ion. Both the prostaglandin H synthase:arachidonic acid and horseradish peroxidase:hydrogen peroxide systems were used to investigate the binding of [3H]-2-AF to DNA

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... d the nature of the DNA adducts formed from peroxidative activation of 2-AF. Modification of DNA by N-hydroxy-2-AF under mildly acidic conditions was used as a reference system in these studies and yielded a single 2-AF:nucleoside adduct, identified as N-(deoxyguanosin-8-yl)-2-AF (C8-dGuo-AF). Enzymatic hydrolysis of DNA modified by 2-AF activated in either of the peroxidase systems liberated 2-AF:nucleoside adducts that differed considerably from C8-dGuo-AF in chromatographic and extraction properties. C8-dGuo-AF from DNA hydrolysates was easily extracted into n-butyl alcohol and adsorbed by Sephadex LH-20 columns. In contrast, the peroxidase-derived adducts were poorly extracted into n-butyl alcohol and were not retained on Sephadex LH-20 columns. Experimental evidence suggests the peroxidase-derived adducts may possess a negative charge at neutral pH. Since C8-dGuo-AF is the only 2-AF:nucleoside adduct formed when 2-AF is activated via N-hydroxylation, these new adducts represent a marker unique to peroxidative activation of 2-AF. Therefore, 2-AF:DNA adducts can be used as a differential end point with which to assess the relative roles of N-hydroxylation and peroxidation in the metabolic activation of 2-AF in cell culture and in target tissues in vivo.