Metabolism of sanguinarine in human and in rat: Characterization of oxidative metabolites produced by human CYP1A1 and CYP1A2 and rat liver microsomes using liquid chromatography–tandem mass spectrometry

Abstract

The quaternary benzo[c]phenanthridine alkaloid, sanguinarine (SA), has been detected in the mustard oil contaminated with Argemone mexicana, which produced severe human intoxications during epidemic dropsy in India. Today, SA metabolism in human and in rat has not yet been fully elucidated. The goal of this study is to investigate the oxidative metabolites of SA formed during incubations with rat liver microsomes (RLM) and recombinant human cytochrome P450 (CYP) and to tentatively identify the CYP isoforms involved in SA detoxification. Metabolites were analyzed by liquid chromatography combined with electrospray ionization-tandem mass spectrometry. Up to six metabolites were formed by RLM and their modified structure has been proposed using their mass spectra and mass shifts from SA (m/z 332). The main metabolite M2 (m/z 320) resulted from ring-cleavage of SA followed by demethylation, whereas M4 (m/z 348) is oxidized by CYP in the presence of NADPH. The diol-sanguinarine metabolite M6 (m/z 366) formed by RLM might derive from a putative epoxy-sanguinarine metabolite M5 (m/z 348). M4 and M6 could be detected in rat urine as their respective glucuronides. 5,6-Dihydrosanguinarine is the prominent derivative formed from SA in cells expressing no CYP. Oxidative biotransformation of SA was investigated using eight human CYPs: only CYP1A1 and CYP1A2 displayed activity.

Introduction

Sanguinarine (SA) is a quaternary benzo[c]phenanthridine alkaloid (Fig. 1a) present in Paperveraceae, particularly in Argemone mexicana L. seed and in the rhizome of Sanguinaria canadensis L. SA toxicity has been evidenced during Epidemic Dropsy (ED) in India resulting from the consumption of mustard oil educorated by Argemone oil. Several outbreaks of dropsy have been reported in 1998 in Delhi. Recently largest outbreak of ED appeared in the country involving over 2992 victims admitted to hospital and more than 67 deaths [1]. Although SA exhibits anti-bacterial and anti-inflammatory properties, in vitro studies using various human cells demonstrate that SA is a toxic compound exhibiting potential antitumor activity, as reported by Karp et al. [2]. SA is shown to bind with rat tubulin to inhibit the microtubule polymerization and can readily intercalate double-stranded DNA, causing DNA single strand breaks. Due to its structural similarity with polycyclic aromatic hydrocarbons, SA might act as a potential procarcinogen. Moreover, aryl hydrocarbon receptor metabolic signalling pathways might modulate SA activity, according to Karp et al. [2]. Recently, in vitro metabolism studies in rat showed that cytochrome P4501A1 (CYP1A1) and P4501A2 (CYP1A2) were involved in SA metabolism. Thus, Vrba et al. [3] report that CYP1A2 could likely modulate SA toxicity. A few toxicological studies of SA have been recently conducted in animals [4]. Williams et al. [5] demonstrate that administration of SA (10 mg/kg mice) results in significant decrease of liver glutathione and CYP enzymes activities. SA is reduced [6] into 5,6-dihydrosanguinarine (DHSA), identified in rat plasma and liver by liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) [7]. A pharmacokinetic study of DHSA [8] shows recently no toxicity in rat using repeated dosing of 58 mg/kg/day. Actually SA toxicity mechanism may be partially explained by the production of reactive oxygen species such as peroxide oxygen [9] generated by enzyme-catalyzed redox cycling between the reduced and oxidized forms of phenanthridine. SA cytotoxicity is probably due to a rapid apoptotic response induced by a glutathione depletion effect [10], as demonstrated also in plasma of ED patients by Babu et al. [11]. Recently, Ansari et al. [12] suggested that antioxidants such as riboflavin might provide protection to ED patients in case of acute toxicity with Argemone.

A combination of complementary approaches is required to identify enzyme(s) responsible for the biotransformation of xenobiotics. Once the enzymes known, prediction may be made concerning drug–drug interactions potentially resulting in clinical alteration of pharmacokinetics. Very sensitive HPLC methods coupled to fluorescence detection [13], [14] or electrospray ionization-mass spectrometry (ESI-MS) [7] are required for quantifying SA in culture medium or in rat plasma or urine. Thus, the biotransformation of SA and chelerythrine (CHEL, Fig. 1b) into dihydro derivatives has been successfully characterized by LC–ESI-MS [7], [15].

Previous in vitro studies [2] indicated that CYP-dependent mono-oxygenations were involved in SA metabolism. These investigations conducted with rat or human liver microsomes suggested modulation by CYP1A. In order to understand the toxicity of SA, its metabolism is studied in vitro using induced RLM. Then, SA incubations were conducted with human recombinant CYP to characterize the human metabolic pathway(s) undergone by SA and to identify the CYP isoforms involved in these oxidative reactions. In the present work, LC–ESI-MS/MS was used allowing the characterization of major and minor oxidative SA metabolites produced by human CYP1A (Fig. 2) and RLM in order to do their structural elucidation in comparison with CHEL metabolites formed by RLM. In vivo SA metabolic experiments in rat were performed in order to provide additional information about SA detoxification pathways.