The role of efflux transporters on the transport of highly toxic aconitine, mesaconitine, hypaconitine, and their hydrolysates, as determined in cultured Caco-2 and transfected MDCKII cells

Abstract

Aconitum alkaloids including aconitine (AC), mesaconitine (MA), hypaconitine (HA), are highly toxic. Their hydrolysates, such as benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine, and mesaconine, are considerably less toxic. Efflux transporters, including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein isoform 2 (MRP2), act as a first line of defence and play key roles in toxicity prevention. The aim of the present study was to determine the role of efflux transporters in the transport of Aconitum alkaloids using cultured Caco-2, MDR1-MDCKII and BCRP-MDCKII cells. Bidirectional transport assays of the Aconitum alkaloids were performed with or without P-gp (cyclosporine A and verapamil), BCRP (Ko143) and MRP2 (MK571) inhibitors. The efflux ratios (Er) of AC, MA, and HA in Caco-2 cells were 34.6 ± 4.2, 29.7 ± 2.1, and 15.6 ± 2.1, respectively; those of BAC, BMA, and BHA were approximately 4, and those of aconine and mesaconine were equal to 1. The Er values of AC, MA, and HA in MDR1-MDCKII and BCRP-MDCKII cells were significantly higher than those in parental MDCKII cells. Taken together the results of Er values and intracellular amounts in the presence of inhibitors, P-gp and BCRP were involved in the transport of AC, MA and HA; and MRP2 might transport AC, MA, HA, BAC, BMA and BHA.

Introduction

For over 2000 years, Aconitum has been applied in a wide range of therapeutic uses, including treatments for joint pain, gastroenteritis, diarrhea, and rheumatoid arthritis (Suzuki et al., 1994). However, the improper use of Aconitum may result in severe intoxication involving cardio- and neurotoxicity (Gutser et al., 1998). The toxicological mechanism of interaction of Aconitum with voltage-dependent Na⁺ channels is widely recognized (Fu et al., 2006). Patients, upon ingestion of Aconitum, may present with nausea, vomiting, arrhythmia, coma, and even death. A male patient was reported to have eaten the blue flowers of Aconitum napellus and died (Pullela et al., 2008). Thus, the clinical use of Aconitum has been prohibited in Europe. However, Aconitum is still widely used in China. In Traditional Chinese Medicine, the herb is used after several hours of decoction. Published studies show that decoction reduces Aconitum toxicity by decomposition of aconitine (AC), mesaconitine (MA), and hypaconitine (HA) into benzoylaconine (BAC), benzolymesaconine (BMA), benzoylhypaconine (BHA), aconine, mesaconine, and hypaconine (see Fig. 1; hypaconine was not included in the present study because of the lack of commercially available standards). The loss of acetyl and benzoyl groups in diester diterpenoid Aconitum alkaloids appears to significantly decrease their toxicity by over 100-fold. The oral median lethal doses (LD₅₀) of the toxins in mice are 1.8 mg/kg AC (Wada et al., 2005), 1.9 mg/kg MA (Singh et al., 1986), 5.80 mg/kg HA (Bisset, 1981), 1500 mg/kg BAC (Wada et al., 2005), 810 mg/kg BMA (Bisset, 1981), and 830 mg/kg BHA (Bisset, 1981).

Efflux transporters and metabolic enzymes in the intestine and liver protect the body by limiting the invasion of xenobiotics, especially toxicants. ATP Binding Cassette (ABC) transporters, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein isoform 2 (MRP2), are abundantly expressed in the apical membranes of intestinal epithelial, hepatic, and renal tubular cells and modulate the absorption, distribution, metabolism, excretion, and toxicity of pharmacological agents. During oral absorption, efflux transporters located in the apical brush border membrane of enterocytes actively cause the efflux of drugs from gut epithelial cells back into the intestinal lumen (Hoffmaster et al., 2004, Kim and Benet, 2004). Efflux transporters play important roles in limiting toxicant absorption to avoid poisoning. The effect of transporter-mediated efflux on intestinal absorption could significantly alter the safety and efficacy of therapy. Thus, characterization of the effect of efflux transporters on the transport of compounds must be seriously considered, especially for toxic drugs. Aconitum has a narrow therapeutic index. Serious toxic effect may occur even if the oral dose of Aconitum is a little bit higher than the therapeutic dose. No publication thus far has reported the effect of efflux transporters on the transport of Aconitum alkaloids, the investigation of which is significant for clinical practice.

The human colonic adenocarcinoma Caco-2 cell line is most widely employed for evaluating drug intestinal transport mechanisms in vitro (Zornoza et al., 2004). Caco-2 membranes also functionally express P-gp, BCRP, and MRP2 (Maeng et al., 2002) and are therefore employed extensively for studying P-gp, BCRP, and MRP-mediated transport (Konsoula and Jung, 2009). Polarized cell monolayers over-expressing P-gp, BCRP and MRP2 have been used extensively as model systems for studying P-gp, BCRP and MRP2 transport mechanisms, respectively (Tran et al., 2005). The Mardin–Darby canine kidney (MDCK) cell is a dog renal epithelial cell line. In the late 1990s, MDR1-MDCKII (MDCKII cell transfected with human MDR1), BCRP-MDCKII (MDCKII cell transfected with human BCRP) and MRP2-MDCKII (MDCKII cell transfected with human MRP2) cell lines have been reported to express high levels of P-gp, BCRP and MRP2 on the apical side (A) of the polarized cell monolayer, respectively. Therefore, the combined use of Caco-2 and transfected MDCKII cells, as well as specific inhibitors of efflux transporters, could yield clear advantages in the study of transport interactions between Aconitum alkaloids and efflux transporters.

In the present study, the bidirectional transport of highly toxic Aconitum alkaloids across Cac-2, MDR1-MDCKII and BCRP-MDCKII cells in the absence and presence of efflux transporters inhibitors was investigated. Evaluations were carried out to determine the effect of efflux transporters on the intestinal transport of the Aconitum alkaloids. The results of the present study could provide significant information for clinical practice.