Original Contribution
α-Tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

https://doi.org/10.1016/j.freeradbiomed.2006.06.022Get rights and content

Abstract

To test the hypothesis that supra-elevated hepatic α-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic α-tocopherol metabolism and excretion, rats received daily subcutaneous α-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver α-tocopherol concentrations increased from 12 ± 1 nmol/g (mean ± SE) to 819 ± 74 (Day 3), decreased at Day 9 (486 ± 67), and continued to decrease through Day 18 (338 ± 37). α-Tocopherol metabolites and their intermediates increased and decreased similarly to α-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic α-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver α-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic α-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.

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

References (54)

  • R.J. Sokol et al.

    Vitamin E deficiency in adults with chronic liver disease

    Am. J. Clin. Nutr.

    (1985)
  • R.J. Sokol et al.

    Mechanism causing vitamin E deficiency during chronic childhood cholestasis

    Gastroenterology

    (1983)
  • R.J. Sokol et al.

    Vitamin E reduces oxidant injury to mitochondria and the hepatotoxicity of taurochenodeoxycholic acid in the rat

    Gastroenterology

    (1998)
  • E. Gumpricht et al.

    Enrichment of rat hepatic organelles by vitamin E administered subcutaneously

    Free Radical Biol. Med.

    (2004)
  • M. Podda et al.

    Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols and ubiquinones

    J. Lipid Res.

    (1996)
  • H. Vaule et al.

    Vitamin E delivery to human skin; studies using deuterated a-tocopherol measured by APCI LC-MS

    Free Radical Biol. Med.

    (2004)
  • S.W. Leonard et al.

    Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration

    J. Lipid Res.

    (2005)
  • M.G. Traber et al.

    Impaired discrimination between stereoisomers of a-tocopherol in patients with familial isolated vitamin E deficiency

    J. Lipid Res.

    (1993)
  • S.W. Leonard et al.

    Incorporation of deuterated RRR- or all rac a-tocopherol into plasma and tissues of a-tocopherol transfer protein null mice

    Am. J. Clin. Nutr.

    (2002)
  • L. Barella et al.

    Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

    Biochim. Biophys. Acta

    (2004)
  • M. Meydani et al.

    Influence of vitamin E supplementaiton on antipyrine clearance in the cebus monkey

    Nutr. Res.

    (1990)
  • M. Murray

    In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

    Biochem. Pharmacol.

    (1991)
  • D.J. Mustacich et al.

    Biliary secretion of alpha-tocopherol and the role of the mdr2 P-glycoprotein in rats and mice

    Arch. Biochem. Biophys.

    (1998)
  • D.J. Mustacich et al.

    Colchicine and vinblastine prevent the piperonyl butoxide-induced increase in rat biliary output of alpha-tocopherol

    Toxicol. Appl. Pharmacol.

    (1996)
  • J.M. Pascussi et al.

    The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors

    Biochim. Biophys. Acta

    (2003)
  • N. Landes et al.

    Vitamin E activates gene expression via the pregnane × receptor

    Biochem. Pharmacol.

    (2003)
  • M.G. Traber et al.

    Vitamin E trafficking

    Ann. N.Y. Acad. Sci.

    (2005)
  • Cited by (0)

    View full text