Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro

Abstract

Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of Hg in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of Cu in both fractions. Fractionation of nuclei following in vivo challenge with Cu showed enrichment of Cu in heterochromatin, relative to euchromatin, while in vivo exposure to Hg resulted in a 20-fold accumulation of Hg in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward Hg and Cu similar to that observed in the in vivo metal challenge experiments. The level of Hg which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 .mu.g of Hg/mg of DNA) to that of in vivo euchromatin-bound Hg after 1 mo. of challenge with dietary metal. Cu showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both Hg and Cu appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of Cu.