Characterization and properties of the DNA adducts formed from N -methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen

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Abstract
Chronic oral administration of the carcinogenic aminoazo dye N -methyl-4-aminoazobenzene (MAB) to rats is known to result in the induction of liver tumors. In order to assess the role of carcinogen-DNA adduct formation in MAB hepatocarcinogenesis, male rats were fed 0.06% [3'- 3 H]MAB in the diet for 1, 3 or 5 weeks. Groups were sacrificed at 0, 24 and 72 h after dosing, and DNA was isolated from the liver and from two non-target tissues, the kidney and spleen. Upon enzymatic hydrolysis of the DNA, [ 3 H]aminoazo dye-nucleoside adduct levels in these tissues were determined by h.p.l.c. Rats concurrently administered unlabeled MAB for 5 weeks and continued on a control diet for 9 months developed hepatocellular carcinomas (16/30 animals). No tumors were observed in 21 rats given only control diets. After chronic administration of [ 3 H]MAB, three major MAB-DNA adducts were found in vivo : N -(deoxyguanosin-8-yl)-MAB (C8-dG-MAB), 3-(deoxyguanosin- N 2 -yl)-MAB (N 2 -dG-MAB) and 3-(deoxyadenosin- N 6 -yl)-MAB (N 6 -dA-MAB). In addition, several minor products were identified as: (i) an (8,9)-purine ring-opened derivative of C8-dG-MAB that may represent an intermediate in DNA repair; (ii) N -guanosin-8-yl-MAB which is present due to trace RNA contamination; (iii) cis isomers of C8-dG-MAB and N -guanosin-8-yl-MAB, formed by photo-illumination during analyses; and (iv) N -(guanin-8-yl)-MAB, a deribosylated product resulting from thermal depurination of C8-dG-MAB. In addition, N -(deoxyguanosin-8-yl)-4-aminoazobenzene (C8-dG-AB), a major adduct previously detected in mouse liver after a single dose of 4-aminoazobenzene, was found in rat liver but appeared to be present in significant amounts only after chronic treatment with MAB. This product co-chromatographed with N 6 -dA-MAB but could be removed by selective decomposition in 0.1 N NaOH. For all tissues examined N 2 -dG-MAB and C8-dG-MAB were the major adducts observed with each accounting for 40-50% of the total carcinogen bound to DNA in rats that were sacrificed immediately after MAB feeding for 1, 3 or 5 weeks. The levels of total MAB-DNA adducts in the liver were 2–10 times greater than in the kidney or spleen and appeared to increase 2- to 3-fold over the dosing period. However, by 24–72 h after cessation of MAB treatment, hepatic C8-dG-MAB showed a rapid decline to levels similar to that found in non-target tissues. The minor adducts, N 6 -dA-MAB and C8-dG-AB, exhibited similar behavior and never accounted for > 5–10% of the total DNA binding. In contrast, hepatic N 2 -dG-MAB was a persistent lesion throughout the treatment regimen; at 72 h after dosing, it accounted for 60–90% of the hepatic DNA adducts and was the only adduct whose levels correlated with target tissue specificity after a complete hepatocarcinogenic dose of MAB.

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