Associations of benzo[ a]pyrene with rat-liver chromatin and the chromatin protein

Abstract

Rat-liver nuclei were prepared in the course of time after the i.p. injection of [G- 3H]benzo[ a]pyrene ([ 3H]BP). The nuclei were lysed in the hypotonic buffer and centrifuged at 4000 × g. The recovery of the radioactivity of resulting supernatant (chromatin) was thus 91% at 24 h, 68% at 48 h and 74% at 168 h after the i.p. injection. The incorporation into nucleosomeoligomer fraction was always much more than into those of monomer and DNA-rich fractions. The preferential incorporation was found in the fraction which was enriched in non-histone chromatin proteins (NHCPs) of 49 000–55 000 daltons. This fraction steadily raised the incorporation level until at 168 h after the i.p. injection. In contrast, the levels of histone and DNA fractions were always very low. The significant incorporation was observed in the fraction which was composed of five classes of histones and low molecular-weight NHCPs (< 30 000 daltons), despite the very low incorporation into the histone fraction. The fluorographic analysis revealed the predominant incorporations at the positions of molecular weight of 65 000, 52 000 and 44 000 daltons. In addition, the incorporations were clearly observed at the positions of 59 000, 49 000, 45 000, H1 histone, A24 protein and another one. On the other hand, these fractions were, at the final preparation steps, subjected to either dialysis or SDS-phenol treatment and/or acetone precipitation. The total recovery of radioactivity was thus 21% at 24 h, 32% at 48 h and 52% at 168 h after the i.p. injection. These results suggest that the chromatin contains considerable amounts of water-soluble, phenol and/or acetone-soluble BP-conjugates in the early period after the i.p. injection.