Murine cytochrome P450 2A5 (CYP2A5) is an interesting enzyme for its unique regulation and its involvement in liver injury caused by various well-known pathological conditions or hepatotoxins. It has been reported that CYP2A5 is upregulated following exposure to chemical hepatotoxins and during pathophysiological conditions in which the levels of most Cytochrome P450s are either unchanged or down-regulated. Recently bilirubin has been identified as the first endogenous substrate for CYP2A5 and it has been suggested that CYP2A5 plays a major role in bilirubin clearance as an alternative mechanism to BR conjugation by UGT1A1. This study investigated the mechanisms of gene regulation and cytoprotective role of CYP2A5 in response to bilirubin treatment in liver. Our results demonstrate that bilirubin induces CYP2A5 expression at the mRNA and protein levels by increasing CYP2A5 transcription via a mechanism that involves Nrf2 activation. Furthermore, our results suggest that induced CYP2A5 plays a cytoprotective role against bilirubin toxicity by directly lowering the cellular levels of bilirubin and by inhibiting caspase-3 activation.iii ACKNOWLEDGEMENTS

Jaundice is a potentially fatal condition resulting from elevated serum bilirubin levels. For centuries, herbal remedies containing Artemisia capillaris Thunb. including the compound 6,7-dimethylesculetin (DE) have been used in Asia to prevent and treat jaundice in neonates. DE activates an important regulator of bilirubin metabolism, the constitutive androstane receptor (CAR), and increases bilirubin clearance. In addition, murine cytochrome P450 2a5 (Cyp2a5) is known to be involved in the oxidative metabolism of bilirubin. Moreover, treatment of mice with phenobarbital, a known inducer of both CAR and Cyp2a5, increases expression of Cyp2a5 suggesting a potential relationship between CAR and Cyp2a5 expression. The aim of this study is to investigate the influence of Artemisia capillaris and DE on the expression and regulatory control of Cyp2a5 and the potential involvement of CAR. Treatment of mouse hepatocytes in primary culture with DE (50 μM) significant increased Cyp2a5 mRNA and protein levels. In mice, Artemisia capillaris and DE treatment also increased levels of hepatic Cyp2a5 protein. Luciferase reporter assays showed that CAR increases Cyp2a5 gene transcription through a CAR response element in the Cyp2a5 gene promoter. Moreover, DE caused nuclear translocation of CAR in primary mouse hepatocytes and increased Cyp2a5 transcription in the presence of CAR. These results identify a potential CAR-mediated mechanism by which DE regulates Cyp2a5 gene expression and suggests that DE may enhance bilirubin clearance by increasing Cyp2a5 levels. Understanding this process could provide an opportunity for the development of novel therapies for neonatal and other forms of jaundice.