Journal Article

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

Cynthia Agus, Kenneth F. Ilett, Fred F. Kadlubar and Rodney F. Minchin

in Carcinogenesis

Volume 21, issue 6, pages 1213-1219
Published in print June 2000 | ISSN: 0143-3334
Published online June 2000 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/21.6.1213
Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

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2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods. These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA. To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters. In this study 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 covalent binding of [3H]N-OH-IQ to DNA. ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity 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-phenylimidazo[4,5-b]pyridine (7.7 ± 0.3 and 0.9 ± 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3′-phosphoadenosine 5′-phosphosulphate and ~50% of that seen with acetyl coenzyme A (AcCoA). In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites. With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, 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 heterocyclic amine-induced carcinogenesis.

Keywords: DMSO, dimethyl sulphoxide; DTT, dithiothreitol; GSH, reduced glutathione; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; N-OH-IQ, N-hydroxy-IQ; N-OH-PhIP, N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine; PKC, protein kinase C; THF, tetrahydrofuran.

Journal Article.  5381 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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