Protopine and allocryptopine increase mRNA levels of cytochromes P450 1A in human hepatocytes and HepG2 cells independently of AhR

Abstract

The isoquinoline alkaloids protopine and allocryptopine are present in phytopreparations from medicinal plants, such as Fumaria officinalis. Since nothing is known about effects of the alkaloids on the expression of xenobiotic-metabolizing enzymes, we examined whether protopine or allocryptopine affect the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells. In HepG2 cells, protopine and allocryptopine significantly increased CYP1A1 mRNA levels after 24 h exposure at concentrations from 25 and 10 μM, respectively, as shown by real-time PCR. Both protopine and allocryptopine also dose-dependently increased CYP1A1 and CYP1A2 mRNA levels in human hepatocytes. However, the effects of the tested alkaloids on both cell models were much lower than the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. Using gene reporter assays performed in transiently transfected HepG2 cells, we demonstrated that the induction of CYP1A1 expression by either protopine or allocryptopine was associated with mild or negligible activation of the aryl hydrocarbon receptor. In contrast to TCDD, CYP1A mRNA levels induced by protopine or allocryptopine in both HepG2 cells and human hepatocytes did not result in elevated CYP1A protein or activity levels as shown by western blotting and EROD assays, respectively. We conclude that the use of products containing protopine and/or allocryptopine may be considered safe in terms of possible induction of CYP1A enzymes.

Introduction

The isoquinoline alkaloids protopine and allocryptopine are mainly found in plants of the Papaveraceae and Fumariaceae families. In biological systems, both alkaloids are considered to exist in equilibrium between their tricyclic bases and tetracyclic quaternary salts (Fig. 1) (Vacek et al., 2010). Protopine and allocryptopine have been the focus of a large number of biological studies in which they both exhibited, for instance, anti-parasitic activity (Satou et al., 2002, Wang et al., 2010) and only weak cytotoxicity in comparison with other types of isoquinoline alkaloids (Satou et al., 2002). Protopine was found to be cytoprotective against oxidative stress-induced cell death in vitro (Xiao et al., 2008). The alkaloid was shown to have anti-arrhythmic (Song et al., 2000), anti-thrombotic, anti-inflammatory (Saeed et al., 1997), and hepatoprotective effects in animal models (Rathi et al., 2008). The biological activity of protopine may be associated with its ability to inhibit calcium (Ko et al., 1992), sodium (Song et al., 2000), and potassium channels (Jiang et al., 2004). Other molecular targets of protopine, as well as those of allocryptopine, remain unknown.

Protopine and allocryptopine are present in human and veterinary phytopreparations from medicinal plants such as Fumaria officinalis, Chelidonium majus and Macleaya cordata (Vacek et al., 2010). Biologically active substances in these phytopreparations could be, and frequently are, proven substrates of biotransformation enzymes. For this reason, research on the alkaloids’ bioactivity is also directed at possible drug-drug interactions that may result from any changes in the expression or activity of cytochromes P450. In humans, the cytochrome P450 (CYP) enzymes comprise a superfamily of mono-oxygenases involved in the biotransformation of both endogenous substrates and xenobiotics, such as drugs, plant secondary metabolites, and environmental pollutants (Anzenbacher and Anzenbacherova, 2001). Some CYP enzymes have also been recognized to play a negative role in chemically induced carcinogenesis. This is, among others, the case for CYP1A1 and CYP1A2 which appear to mediate metabolic activation of a number of procarcinogens, including polycyclic aromatic hydrocarbons (Shimada and Fujii-Kuriyama, 2004). Protopine is metabolized by demethylenation of its methylenedioxy group followed by conjugation reactions. Inhibition studies on rat liver microsomes have revealed that the demethylenation of protopine is catalysed by CYP enzymes CYP2C11, CYP2D1, and to a lesser extent by CYP3A2 and CYP1A2 (Paul et al., 2004). Protopine and allocryptopine have been shown to inhibit several human CYP enzymes. Both alkaloids were reported to be potent inhibitors of CYP2D6 and to suppress, albeit with less potency, the activities of CYP3A4, CYP1A2, and CYP2B6 as well. This aside, protopine and allocryptopine were found to inhibit CYP2C19 and CYP2C8, respectively (Salminen et al., 2010). In contrast, the effects of protopine and allocryptopine on the expression of xenobiotic-metabolizing enzymes have not been assessed to date. In this study, we examined whether protopine and/or allocryptopine affect the expression of CYP1A enzymes in primary cultures of human hepatocytes and human hepatoma HepG2 cells.