It has been reported that Zn7-metallothionein (MT), contains one weak binding site for Zn2+. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn2+. Contrary to the previous study, no evidence was found for Zn2+ stoichiometically bound to the protein with an apparent stability constant of about 108. Indeed, stability constant measurements based upon competition between Zn7-MT and ligands of known stability with Zn2+ showed that all of the protein bound Zn2+ displayed the same stability constant at pH 7.4 and 25° C of (1.7±0.6) × 1011. Brief reaction of Zn7-MT with strong acid converted it into MT* and upon reneutralization into Zn7-MT*, which demonstrated reactivity of about 1 Zn2+/mol MT with competing ligands. Acid titration of Zn7-MT to pH 2 or below rapidly resulted in the formation of Zn7-MT* that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn2+ between pH 5 and 7. Since MT is commonly acidified during preparation, care must be taken to document which form of the protein is present in subsequent experiments at pH 7.
Variability in drug-metabolizing enzyme developmental trajectories contributes to interindividual differences in susceptibility to chemical toxicity and adverse drug reactions, particularly in the first years of life. Factors linked to these interindividual differences are largely unknown, but molecular mechanisms regulating ontogeny are likely involved. To evaluate chromatin structure dynamics as a likely contributing mechanism, age-dependent changes in modified and variant histone occupancy were evaluated within known CYP3A4 and 3A7 regulatory domains. Chromatin immunoprecipitation using fetal or postnatal human hepatocyte chromatin pools followed by quantitative polymerase chain reaction DNA amplification was used to determine relative chromatin occupancy by modified and variant histones. Chromatin structure representing a poised transcriptional state (bivalent chromatin), indicated by the occupancy by modified histones associated with both active and repressed transcription, was observed for CYP3A4 and most 3A7 regulatory regions in both postnatal and fetal livers. However, the CYP3A4 regulatory regions had significantly greater occupancy by modified histones associated with repressed transcription in the fetal liver. Conversely, some modified histones associated with active transcription exhibited greater occupancy in the postnatal liver. CYP3A7 regulatory regions also had significantly greater occupancy by modified histones associated with repressed transcription in the fetus. The observed occupancy by modified histones is consistent with chromatin structural dynamics contributing to CYP3A4 ontogeny, although the data are less conclusive regarding CYP3A7. Interpretation of the latter data may be confounded by celltype heterogeneity in the fetal liver.
The study objective was to evaluate changes in histone marks and the associated changes in chromatin structure as a mechanism controlling drug metabolizing enzyme ontogeny. The relative enrichment of histone marks was determined using chromatin immunoprecipitation with pooled chromatin isolated from 1st trimester primary human fetal or adult hepatocytes with quantitation by RT‐PCR. CYP3A4 expression increases at least 20‐fold between the third trimester and the adult (mean ± SD = 4.5 ± 2.1 versus 89.6 ± 64.0 pmol/mg microsomal protein, respectively). Evaluating the CYP3A4 CLEM4 domain located from position −11,420 to −10,883 and containing 3 USF1, 2 HNF4α, 1 HNF1α, and 1 AP1 elements revealed a 6‐ to 14‐fold enrichment of the H3K27me3 histone mark in fetal versus adult hepatocytes. In contrast, a 1.5‐ to 3‐fold enrichment of the H3K4me1 histone mark was observed in adult versus fetal hepatocytes. A 100‐fold difference in USF1 enrichment was observed in adult versus fetal hepatocytes. Similar results were observed for the presence of these same histone marks and the binding of C/EBPβ in the regulatory domain located between CYP3A4 position −5900 and −5700. Given the association of H3K27 and HeK4me1 with condensed and open chromatin, respectively, tThese data are consistent with changes in chromatin structure having a dominant role in regulating CYP3A4 ontogeny. Supported in part by PHS grant GM081344.
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