ARYL-HYDROCARBON RECEPTOR-DEPENDENT INDUCTION OF CYP1A1 BY BILIRUBIN IN MOUSE HEPATOMA HEPA 1C1C7 CELLS

Heme metabolism normally involves enzymatic conversion to biliverdin a nd subsequently to bilirubin, catalyzed by heme oxygenase and biliverd in reductase, respectively. We examined the ability of exogenously add ed hemin, biliverdin, or bilirubin to regulate Cyp1a1, an enzyme that may be active in bilirubin elimination. A substantial dose-dependent i ncrease in Cyp1a1 mRNA occurred after treatment of Hepa 1c1 c7 cells w ith either of the three compounds. This increase was readily apparent 1 hr after treatment with biliverdin or bilirubin but required greater than or equal to 2 hr with hemin. Treatment of Hepa 1c1c7 cells with these compounds also caused a dose-dependent increase in Cyp1a1-depend ent 7-ethoxyresorufin-O-deethylase (EROD) activity. Of the three compo unds, bilirubin produced the greatest maximal increase in Cyp1a1 mRNA and EROD (5.5-, 10.5-, and 15-fold for 100 mu M hemin, biliverdin, and bilirubin, respectively) activity. The RNA polymerase inhibitor actin omycin D completely blocked Cyp1a1 induction by these compounds, indic ating a requirement for de novo RNA synthesis via transcriptional acti vation. The protein synthesis inhibitor cycloheximide did not affect C yp1a1 mRNA induction, indicating a lack of requirement for labile prot ein factors. In contrast, EROD induction by hemin, biliverdin, or bili rubin was completely blocked by cycloheximide treatment, indicating th at the increase in enzyme activity is dependent on increased Cyp1a1 ap oprotein synthesis. Aryl hydrocarbon receptor (AHR)- and AHR nuclear t ranslocator-deficient mutant Hepa 1c1c7 cells did not exhibit increase d Cyp1a1 mRNA or EROD activity after treatment with these compounds, i ndicating the requirement for a functional AHR for this response. Cons istent with this, hemin, biliverdin, and bilirubin were able to induce expression of the dioxin-response element/luciferase reporter plasmid pGudLuc1.1 after transient transfection into wild-type Hepa 1c1c7 cel ls. Gel retardation assays demonstrated that bilirubin, but not hemin or biliverdin, was able to transform the AHR to a form capable of spec ifically binding to a P-32-labeled oligonucleotide containing a dioxin -response element sequence. These data indicate that bilirubin induces Cyp1a1 gene transcription through direct interaction with the AHR. In contrast, hemin and biliverdin seem to induce Cyp1a1 indirectly by se rving as precursors to the endogenous formation of bilirubin via norma l heme metabolism pathways. This is the first direct demonstration tha t the endogenous heme metabolite bilirubin can directly regulate Cyp1a 1 gene expression and enzymatic activity in an AHR-dependent manner.