Quantitative and Qualitative Characterization of Aflatoxin B1 Adducts Formed in Vivo within the Ribosomal RNA Genes of Rat Liver DMA1

We have examined the time course and patterns of covalent aflatoxin B^DNA adducts produced within the ribosomal RNA gene sequences isolated from the liver nuclear DNA of aflatoxin Bi-treated animals. Liver nuclear DNA was initially enriched in ribosomal DNA by cesium salt density centrifugaron, and incu bated under alkaline conditions to stabilize bound aflatoxin ST ONA moieties. Alkali-treated DNA was hybridized to 188 and 28S rRNA, and the RNAiDNA hybrids were recovered by cesium chloride centrifugation. Ribosomal DNA sequences within nu clear DNA were found to be preferential targets for aflatoxin B! modification. Over a 12-h period after administration of 1-mg [3H]aflatoxin Bi/kg dose ribosomal DNA contained 4 to 5 times more aflatoxin Bi residues per mg DNA than did total nuclear DNA. Aflatoxin B, residues bound to ribosomal DNA were also found to be removed more rapidly than from total nuclear DNA by a factor of 5.7 over the 12-h period postdosing. Levels of the principal aflatoxin B, adduct, 2,3-dihydro-3-hydroxy(A/7guanyl)aflatoxin B,, as well as the stable formamidopyrimidine derivatives of the parent adduct were also determined. Nuclear DNA isolates were heated to induce depurination of the principal N7-guanine adduct, and differences in adduct levels between alkali-treated (stabilized) and depurinated DNA samples were taken as an approximation of initial levels of this aflatoxin B, :DNA moiety in ribosomal isolates. No differences in the proportions of these aflatoxin B,:DNA adduct species were found in ribosomal as compared to total nuclear DNA, and we conclude that the preferential formation and removal of aflatoxin Bi:DNA moieties within ribosomal DNA is not associated with a pattern of adducts qualitatively different from that in total nuclear DNA.