Phenobarbital Induction of CYP2B1/2 in Primary Hepatocytes: Endocrine Regulation and Evidence for a Single Pathway for Multiple Inducers

Abstract

Phenobarbital (PB) and many structurally unrelated chemicals induce the protein and mRNA of P450 cytochromes CYP2B1, CYP2B2, CYP3A1, and specific phase II enzymes to a greater extent in Fischer 344 (F344) than in Wistar Furth (WF) female rats. This sex- and strain-dependent polymorphism can be partly attributed to suppressive effects of thyroid hormone (TH) on WF but not F344 females. We show here that this strain difference was largely retained in primary hepatocyte cultures and could be resolved into two components; (1) Expression of PB-inducible genes—WF hepatocytes had inherently lower basal and PB-induced levels of CYP2B1/2B2 protein and mRNA and UDPGT mRNA; and (2) TH sensitivity—in WF hepatocytes, PB induction, but not basal expression, of CYP2B1/2B2 was three- to fivefold more susceptible to inhibition by TH when the hormone was added to the medium. This second component explains the selective effect ofin vivotreatment with methimazole, which lowers circulating TH and partially improves PB induction in WF female rats. Following transfection of a reporter construct containing a PB-responsive unit (PBRU), the plasmid was activated by PB to similar extents in hepatocytes from both rat strains. TH treatment did not inhibit PB-mediated induction of the plasmid in either cell type. Thus, neither of the components determining the strain polymorphism are linked totrans-activating factors contributing to this PBRU activity. The PB-like inducers, 2,2′,4,4′,5,5′-hexachlorobiphenyl (HCB) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (o,p-DDD), proportionally induced the CYP2B1/2B2 and UDPGT genes and activated the plasmid (HCB = PB > DDD). CYP2B1/2B2 expression following induction by PB and HCB was subject to identical patterns of inhibition by okadaic acid, cAMP, and GH. Together, these data suggest that PB-like inducers utilize the same polymorphic pathway to affect the same PBRU-activating factors.