Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic an d carcinogenic heterocyclic amines formed during the cooking process of pro tein-rich foods, These compounds are highly mutagenic and have been shown t o produce tumours in various tissues in rodents and non-human primates. Met abolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of for ming adducts with DNA, To date, acetylation and sulphation have been propos ed as important pathways in the formation of N-hydroxy esters, In this stud y we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by cova lent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ w as greatest in the cytosolic fraction of rat liver, although significant ac tivity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimid azo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respec tively). Using reported intracellular concentrations of cofactor, the abili ty of ATP to support DNA binding was similar to that seen with 3'-phosphoad enosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coen zyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction m ixtures using ATP as co-factor showed the presence of two stable, polar met abolites, With AcCoA, only one metabolite was seen. The kinase inhibitors g enistein, tyrphostin A25 and rottlerin significantly inhibited both DNA bin ding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhib itor staurosporine had no effect and the inactive analogue of genistein, da idzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of het erocyclic amine-induced carcinogenesis.