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Quantitative and Qualitative Characterization of Aflatoxin B₁ Adducts Formed in Vivo within the Ribosomal RNA Genes of Rat Liver DNA¹

Laboratory of Toxicology, Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

We have examined the time course and patterns of covalent aflatoxin B₁:DNA adducts produced within the ribosomal RNA gene sequences isolated from the liver nuclear DNA of aflatoxin B₁-treated animals. Liver nuclear DNA was initially enriched in ribosomal DNA by cesium salt density centrifugation, and incubated under alkaline conditions to stabilize bound aflatoxin B₁-DNA moieties. Alkali-treated DNA was hybridized to 18S and 28S rRNA, and the RNA:DNA hybrids were recovered by cesium chloride centrifugation. Ribosomal DNA sequences within nuclear DNA were found to be preferential targets for aflatoxin B₁ modification. Over a 12-h period after administration of 1-mg [³H]aflatoxin B₁/kg dose ribosomal DNA contained 4 to 5 times more aflatoxin B₁ residues per mg DNA than did total nuclear DNA. Aflatoxin B₁ 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 B₁ adduct, 2,3-dihydro-3-hydroxy(N⁷-guanyl)aflatoxin B₁, 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 B₁:DNA moiety in ribosomal isolates. No differences in the proportions of these aflatoxin B₁:DNA adduct species were found in ribosomal as compared to total nuclear DNA, and we conclude that the preferential formation and removal of aflatoxin B₁:DNA moieties within ribosomal DNA is not associated with a pattern of adducts qualitatively different from that in total nuclear DNA.

¹ Financial support for this work was provided by Grants 5-T32-E507020 and 5-PO1-ES00597 from NIH.

² Present address: Laboratory of Toxicology, Department of Veterinary Anatomy, Texas A & M University, College Station, TX 77843.

³ To whom requests for reprints should be addressed.

Received 12/26/84. Revised 4/19/85. Accepted 4/24/85.