Original Contributionα-Tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats
Introduction
Plants synthesize eight different molecules with vitamin E antioxidant activity, consisting of α-, β-, γ-, and δ-tocopherols and the corresponding four tocotrienols. Only α-tocopherol, not the others, is maintained in human plasma and tissues, as a result of the function of the hepatic α-tocopherol transfer protein (α-TTP) [1]. Unlike other fat-soluble vitamins, vitamin E is not accumulated in the body to toxic levels, suggesting that mechanisms, i.e., metabolism and excretion, prevent excess accumulation [2]. “Simon metabolites” were the first vitamin E metabolites described [3]; however, modern techniques to prevent in vitro oxidation have since shown that Simon metabolites occur largely from poor sample handling [2] and that the biologically relevant metabolites of vitamin E are CEHC (2′-carboxyethyl-6-hydroxychroman) products of the respective forms of vitamin E. CEHCs were first described in rats injected with δ-tocopherol [4]. The major metabolite of α-tocopherol, α-CEHC (2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman), is found in the liver, urine, plasma, and bile [5], [6].
The proposed pathway of metabolism of α-tocopherol to α-CEHC (Fig. 1) is based on data from in vitro studies in which intermediate metabolites were isolated and identified from HepG2 cells and rat liver subcellular fractions incubated with various forms of vitamin E [7]. The pathway includes an initial ω-oxidation of the side chain catalyzed by the cytochrome P450 (CYP) system. Ketoconazole and sesamin, known inhibitors of CYP activity, have been shown to inhibit tocopherol metabolism in cell culture studies, while stimulation with rifampicin, an inducer of CYP3A, increased tocopherol metabolism [8], [9], [10], [11], thus supporting the hypothesis that CYP enzymes are required for tocopherol metabolism. In studies using insect microsomes expressing recombinant human CYP enzymes, CYP4F2 metabolized γ-tocopherol and, to a much lesser extent, α-tocopherol, to their respective 13′-OH-tocopherol metabolites [7]. The formation of 13′-OH-α-tocopherol is followed by several steps of β-oxidation leading to the formation of 5′-α-CMBHC (2,5,7,8-tetramethyl-2-(4′-carboxy-4′-methylbutyl)-6-hydroxychroman) and finally α-CEHC (Fig. 1). Although this metabolic pathway has been studied and the intermediate metabolites identified using γ-tocopherol as a model substrate [7], to our knowledge, there are no reports quantifying intermediate metabolites of α-tocopherol metabolism in vivo.
In addition to metabolizing vitamin E, the hepatic CYP enzyme system is responsible for the metabolism of numerous endogenous and exogenous compounds, including the majority of pharmaceutical drugs [12]. The human genome encodes approximately 57 CYP proteins that make up 18 families and 43 subfamilies; however, members of the CYP3A, CYP2B, and CYP2C subfamilies are responsible for the metabolism of most pharmaceutical drugs [12]. The CYP3A subfamily alone is responsible for the metabolism of approximately 50% of all pharmaceutical drugs, as reviewed [13]. Thus dietary supplements that alter one or more of these three CYP subfamilies can have potent adverse effects on drug efficacy.
We have recently shown in mice that hepatic Cyp3a protein levels are increased in mice fed an α-tocopherol-sufficient diet (31 mg/kg diet) compared to mice fed an α-tocopherol-deficient diet (< 2 mg/kg diet), indicating that hepatic Cyp3a protein levels correlate with hepatic α-tocopherol levels [14]. Similarly, hepatic Cyp3a11 mRNA was increased in mice fed a 20 mg α-tocopherol/kg diet as compared to mice fed a 2 mg α-tocopherol/kg diet and was further increased in mice maintained for 9 months on a diet containing 200 mg α-tocopherol/kg [15]. These data suggest that vitamin E supplements might alter human drug metabolism. Moreover, recent reports from clinical trials suggest possible nutrient–drug interactions between α-tocopherol and pharmaceutical drugs metabolized by CYP enzymes, i.e., cholesterol lowering agents such as simvastatin [16], [17]. In addition, MDR1 (multidrug resistance protein 1, also ABCB1 or p-glycoprotein), one of the ABC transporter proteins located in the canalicular membranes of hepatocytes, shares many of the same substrates with CYP3A [18]. Importantly, compounds that modulate CYP3A have been observed to similarly modulate MDR1 [18]. MDR1 is responsible for the biliary excretion of numerous pharmaceutical drugs and confers a multidrug resistance phenotype to cancer cells [19]. α-Tocopherol has been shown to antagonize the ability of MDR1 substrates, such as verapamil, to reverse MDR1-mediated drug resistance in a lung cancer cell line [20]. However, α-tocopherol modulation of the expression of this or other biliary transporters in vivo has not been reported.
The tolerable upper intake level (UL) of RRR-α-tocopherol in humans has been set at 1000 mg/day [21] and was determined based on studies in rats that showed a lowest observed adverse effect level (LOAEL) of 500 mg/kg body wt/day using hemorrhagic effects as the end point for toxicity [21]. Bioavailability of orally administered α-tocopherol is limited by intestinal absorption with the percentage of α-tocopherol absorbed decreasing with increasing oral dose [22], [23]. The bioavailability of oral α-tocopherol is even poorer in patients with cholestatic liver disease, which is unfortunate as in vitro studies indicate that α-tocopherol may significantly reduce the hepatocellular injury associated with this disease [24], [25], [26]. Subcutaneous injections of pharmacologic doses of α-tocopherol are being investigated as a practical long-term route of administration, as compared to parenteral administration, for achieving therapeutic levels of hepatic α-tocopherol in patients with cholestatic liver disease [27]. However, the effects of high doses of α-tocopherol given subcutaneously have not been determined with respect to altered α-tocopherol metabolism or hepatic xenobiotic pathways.
Based on the in vitro and mouse Cyp3a data detailed above, we hypothesized that α-tocopherol up-regulates hepatic xenobiotic pathways, including those involving metabolism and excretion, to prevent its own overaccumulation. To test this hypothesis, using a subcutaneous dosing regiment in rats relevant to that proposed for use in human studies and which is below the LOAEL (rats), we investigated the ability of supra-elevated hepatic α-tocopherol concentrations to concurrently alter α-tocopherol metabolism, as well as alter the expression of hepatic CYP enzymes, α-tocopherol transfer protein, and biliary ABC transporters.
Section snippets
Reagents
Vital E-300 is a nonaqueous injectable form of d-α-tocopherol containing 300 IU RRR-α-tocopherol/ml compounded with 20% ethanol and 1% benzyl alcohol in an emulsified base (Schering-Plough Animal Health, Union, NJ). HPLC-grade methanol, hexane, ethanol, and glacial acetic acid were obtained from Fisher (Fair Lawn, NJ). Antibodies were obtained as follows: anti-rat CYP3A2 and anti-actin (Chemicon, Temecula, CA), anti-human MDR1 (C219 and C494, Signet, Dedham, MA), anti-rat α-TTP (a gift from R.
Tissue and serum α-tocopherol and α-CEHC measurements
α-Tocopherol was administered daily to rats by subcutaneous injection (10 mg/100 g body wt) starting on Day 1. Daily subcutaneous (sc) injections of rats with vehicle did not alter the serum or hepatic α-tocopherol levels as compared with untreated Day 0 rats [27], [34]. Liver α-tocopherol concentrations on Day 0 were 11.7 ± 1.4 nmol/g (mean ± SE), and then at Day 3 were increased about 75-fold (819 ± 74 nmol/g, P < 0.001, Day 3 compared with Day 0, Fig. 2A) and remained elevated through Day 6 (831.4 ±
Discussion
Our data shows that pharmacologic doses of α-tocopherol initially result in significantly increased hepatic levels of α-tocopherol; however, despite continued daily injections of α-tocopherol, hepatic levels of α-tocopherol began to decrease, suggesting that “excess” α-tocopherol was being eliminated from the liver. Data from studies of patients with genetic α-TTP defects [35], [36], as well as studies with Ttpa–/– mice [28], [37], have demonstrated that the hepatic α-TTP is responsible for the
Acknowledgments
This work was supported by a grant to M.G.T. (NIH DK59576), grants to R.J.S. (NIH DK38446 and the Madigan Foundation), and the Environmental Health Sciences Center at Oregon State University (NIEHS P30 ES00210). The Natural Source Vitamin E Association provided partial support for the purchase of the LC-MS. Standards of 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC) and 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC) (LLU-α) were gifts from Dr. William Wechter
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