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Nuclear Distribution of Aflatoxin B₁ and Its Interaction with Histones in Rat Liver in Vivo¹

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

The distribution and retention of [³H]aflatoxin B₁ (AFB₁) residues in rat liver nuclear fractions were determined at 0.5, 2, 12, and 36 hr following administration of a single i.p. dose of [³H]AFB₁ (1 mg/kg body weight). Approximately 85 to 90% of the nuclear-bound AFB₁ was associated with chromatin 2 hr after aflatoxin treatment, with the remainder present in the nucleoplasmic fraction. Further fractionation of the chromatin showed that approximately 80% of the aflatoxin residues was bound to the DNA, 10% was removed by dialysis, and the remaining 10% was recoverable in the dialyzed chromatin protein fraction. The apparent rate of removal of aflatoxin residues from DNA was twice as fast (t_1/2 = 12 hr) as that from the chromatin proteins (_1/2 = 24 hr).

Liver nucleoplasmic and chromatin proteins were separated by sodium dodecyl sulfate:polyacrylamide gel electrophoresis. No major differences were observed in the Coomassie Blue dye-binding patterns of control and AFB₁-treated animals for at least 36 hr postdosing. Although no significant radiolabeled protein peaks were detected in the nucleoplasmic fraction, two major peaks, accounting for 50 to 60% of the total activity applied to the gel, were observed at each time point in the chromatin protein fraction, with apparent molecular weights of 15,000 and 30,000. Approximately 5 to 10% of the total nuclear-bound aflatoxin residues were associated with these proteins, which were identified as histones by their extraction in 0.25 N HCI and their fractionation patterns on polyacrylamide gel filtration columns and acid:urea gel electrophoresis.

Specific activity measurements demonstrated that histone H1 was the major protein target for [³H]AFB₁ residue binding with an adduction level 3 to 4 times higher than that for any other histone. A linear, dose-dependent relationship for the binding of [³H]AFB₁ residues to DNA, histone H1, and the total histone population was noted over a 16-fold dose range (0.125 to 2.0 mg AFB₁ per kg).

Turnover rates of rat liver histones as a function of AFB₁ exposure were determined by labeling the amino acids of histones by i.p. injection of sodium [¹⁴C]bicarbonate 22 hr prior to treatment with the toxin and sacrificing the animals up to 4 days following the dosage protocol. The half-life of histone H1 and the total histone population was identical (approximately 3.5 days) in both control and AFB₁-treated rats. However, the rate of removal of [³H]AFB₁ residues following either a 0.125-or a 1.0-mg/kg dose was 3 to 4 times faster (t_1/2 = approximately 1 day) than was the decrease in specific activity of the ¹⁴C-labeled histone backbone.

¹ Part of this work was presented at the 1979 Annual Meeting of the American Association for Cancer Research (22). Financial support was provided by Grant 5 P01 ES 00597 from the NIH.

² Present address: Laboratory of Experimental Pathology, Building 37-3A07, National Cancer Institute, NIH, Bethesda, Md.

Received 2/12/80. Accepted 8/15/80.