Regulation of cytochromes P450 1A and 2B expression by 4-n-alkyl-methylenedioxybenzenes
Abstract
The role of the Ah receptor in the mechanism(s) by which the family of naturally occurring methylenedioxybenzene (MDB) compounds regulate CYP1A1 expression has been unclear. A series of 4-n-alkyl-substituted MDBs (n = 0-8) were synthesized and utilized to elucidate the molecular mechanisms of MDB-mediated regulation of CYP1A1 expression. Mouse hepatoma, Hepa-1, cells and its mutants BP$\sp{\rm r}$c1 (Arnt-deficiency) and BP$\sp{\rm r}$c4 (AhR deficient) cells were utilized as cells models. A dose- and structure-dependent increase in CYP1A1 apoprotein and holoenzyme were observed in wild-type but not mutant cells where longer chain MDB analogs were more effective inducers of CYP1A1 expression. These results correlated with in vivo data in Sprague-Dawley rat liver as well as in vitro data utilizing a rat hepatoma cell line, H4IIE. Ah receptor (AhR) ligand binding antagonists $(\alpha$-naphthoflavone $\lbrack 1 \mu\rm{M}\rbrack$ or 2,2$\sp\prime,5,5\sp\prime$-PCB (60 $\mu$M)) blocked increases in CYP1A1 expression in wild-type cells. Time-course studies showed a peak in CYP1A1 mRNA at 1 hour post treatment with 6 $\mu$M $\beta$NF or 100 $\mu$M C$\sb6$MDB. MDBs stimulated expression of a DRE-driven firefly luciferase reporter gene in Hepa-1 cells in a dose- and structure-dependent manner. Gel retardation analysis (GRA) revealed that MDBs can activate binding of cytosolic AhR to consensus DRE sequences in a dose- and structure-dependent manner, and sucrose density gradient assays indicated that the longer chain MDB analogs can compete with TCDD for AhR binding. These results indicate that MDBs regulate expression of CYP1A1 via an Ah receptor dependent mechanism involving binding of MDB-Ah receptor complex to responsive elements upstream of the CYP1A1 gene. Along with CYP1A1, MDBs regulate expression of members of the CYP2B family. In Western blots from MDB-treated Sprague-Dawley rat livers the longer chain MDB analogs induced the appearance of a third immunologically related CYP2B novel isozyme. This novel CYP2B isozyme migrated slower in polyacrylamide gels and crossreacted with an antibody specific to a CYP2B2 splicing variant previously reported. In conclusion 4-n-alkyl-MDBs regulate CYP450 expression by several mechanisms: transcriptional (CYP1A1); post-transcriptional (CYP2B2 and CYP2B2v); and post-translational (CYP1A2 and CYP2B2).
Degree
Ph.D.
Advisors
Marcus, Purdue University.
Subject Area
Pharmacology|Molecular biology
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