Activation of the aryl hydrocarbon receptor by berberine in HepG2 and H4IIE cells: Biphasic effect on CYP1A1 Radim Vrzal a , Ade ´la Zdarˇilova ´ a , Jitka Ulrichova ´ a , Lude ˇk Bla ´ha b , John P. Giesy c , Zdene ˇk Dvor ˇa ´k a, * a Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky ´ University, Hne ˇvotı ´nska ´ 3, 77515 Olomouc, Czech Republic b Masaryk University Brno, Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Kamenice 3, 62500 Brno, Czech Republic c Michigan State University, 218c National Food Safety and Toxicology Center, East Lansing, MI 48824, USA Received 23 May 2005; accepted 20 June 2005 Abstract Berberine has long been considered a candidate for an antimalarial drug. It exerts a plethora of biological activities and has been used in the treatment of diarrhea and gastro-enteritis for centuries. Here we provide evidence that berberine activates the aryl hydrocarbon receptor (AhR) in human hepatoma (HepG2) and rat hepatoma cells stably transfected with a dioxin responsive element fused to the luciferase gene (H4IIE.luc). AhR was activated by high doses of berberine (10–50 mM) after 6 and 24 h of incubation as revealed by CYP1A1 mRNA expression (HepG2) and AhR-dependent luciferase activity (H4IIE.luc). Berberine induced nuclear translocation of AhR-GFP chimera transiently transfected to Hepa1c1c7 cells. In contrast, low doses of berberine (<1 mM) and prolonged times of the treatments (48 h) failed to produce any activation of AhR in H4IIE.luc cell line. HPLC analysis ruled out the hypothesis that the loss of berberine capacity to activate AhR in H4IIE.luc cells is due to metabolic inactivation of the alkaloid. We demonstrate that berberine is a potent inhibitor (IC 50 = 2.5 mM) of CYP1A1 catalytic activity (EROD) in HepG2 cell culture and in recombinant CYP1A1 protein. Collectively, our results imply that while berberine activates the Ah receptor, it is accompanied by inactivation of the catalytic activity of CYP1A1 and occurs at concentrations that exceed those predicted to occur in vivo. Given these data, it appears that activation of the AhR pathway by berberine has a low toxicological potential. # 2005 Published by Elsevier Inc. Keywords: Berberine; Metabolism; AhR; Cytochrome P450 1. Introduction Berberine is a quaternary isoquinoline alkaloid found in many different plants e.g. Berberis aristata DC., Berberis vulgaris L., Hydrastis canadensis L. (goldenseal), Coptis chinensis. Due to its antimicrobial, antimotility, and anti- secretory activity, berberine-containing extracts were used as a remedy for the treatment of diarrhea and gastro- enteritis for centuries [1]. Berberine has also been exten- sively studied as a promising antimalarial drug [2,3].A variety of berberine biological effects, related to its anti- inflammatory activity, for instance inhibition of trinitro- benzene sulfonic acid-induced colitis in rats [4] and pre- vention of cyclophosphamide-induced cystitis [5], have been described. Berberine inhibits gene expression of prostaglandin H synthase, a key enzyme in the inflamma- tory response of the organism [6]. At the molecular level, berberine exerts several other effects, such as inhibitory activity on potassium channels [7] and on DNA strand cleavage [8]. In recent years, products of natural origin are being used to a growing extent in both rational pharma- cotherapy and as dietary supplements. Due to cytochrome www.elsevier.com/locate/biochempharm Biochemical Pharmacology 70 (2005) 925–936 Abbreviations: AhR, aryl hydrocarbon receptor; AhR-GFP, chimera aryl hydrocarbon receptor-green fluorescent protein; ARNT, AhR nuclear translocator; CAR, constitutive androstane receptor; CDK, cyclin-depen- dent kinase; COX-2, prostaglandin H synthase; DRE, dioxin-responsive element; ERK, extracellular signal-regulated kinase; EROD, 7-ethoxyre- sorufin-O-deethylase; GR, glucocorticoid receptor; JNK, Jun-N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; PXR, pregnane X receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p- dioxin; VDR, Vitamin D receptor * Corresponding author. Tel.: +420 58 5632324; fax: +420 58 5632302. E-mail address: moulin@email.cz (Z. Dvor ˇa ´k). 0006-2952/$ – see front matter # 2005 Published by Elsevier Inc. doi:10.1016/j.bcp.2005.06.016