Plants synthesize eight different molecules with vitamin E antioxidant activity, consisting of α-, β-, δ-, and γ-tocopherols and the corresponding four tocotrienols. Only α-tocopherol, not the others, is preferentially maintained in human plasma and tissues, as a result of the function of the hepatic α-tocopherol transfer protein (α-TTP) and increased metabolism of non-α-tocopherols relative to α-tocopherol (1
). Unlike other fat-soluble vitamins, α-tocopherol is not accumulated in the body to toxic levels, suggesting that metabolism and excretion, are up-regulated to prevent excess α-tocopherol accumulation (2
Hepatic enzymes and transporters responsible for the metabolism and excretion of various xenobiotic compounds are categorized in three phases. Phase I consists mainly of the cytochrome P450 (CYP) superfamily of enzymes responsible for the oxidation of numerous compounds, such as drugs, vitamins and environmental toxicants, thereby preparing them for conjugation by Phase II enzymes (3
). Phase II enzymes include, but are not limited to, sulfotransferase (SULT), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) superfamilies. The SULTs and UGTs catalyze sulfation and glucuronidation, respectively, of compounds with a hydroxyl group, or once a hydroxyl group has been added following its biotransformation by Phase I enzymes (4
). Key Phase III members are the ATP-binding cassette (ABC) superfamily of transporters that are responsible for excretion of xenobiotic compounds and/or their metabolites from the liver. ABC transporters expressed on hepatic canalicular membranes are the multidrug resistance (MDR) proteins, MDR1 (ABCB1, P-glycoprotein) and MDR3 (ABCB4), the multidrug resistance-related proteins 2 and 6 (MRP2, ABCC2; MRP6, ABCC6) and the breast cancer resistance protein (BCRP, ABCG2), as reviewed (6
The proposed pathway of α-tocopherol metabolism, including an initial ω-oxidation catalyzed by the CYP system to form 13′-OH-α-tocopherol, was initially based on data from in vitro studies in which intermediate metabolites were isolated and identified from HepG2 cells and rat liver sub-cellular fractions incubated with various forms of vitamin E (7
). Additional cell culture studies have shown that inhibitors of CYP activity inhibit tocopherol metabolism, thereby supporting the hypothesis that CYP enzymes are required for tocopherol metabolism. The formation of 13′-OH-α-tocopherol is followed by several steps of β-oxidation leading to the formation of α-CEHC (2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman), the major metabolite of α-tocopherol. α-CEHC is found in the liver, urine, plasma and bile (8
) in the free form and as either a sulfate or glucuronide conjugate (2
), thus suggesting a role for SULTs and UGTs in tocopherol disposition. Moreover Mdr2 (the mouse equivalent of rat and human MDR3), plays a key role in biliary α-tocopherol excretion (13
). And MDR1 was found to be increased by elevated liver α-tocopherol (14
). The role of various hepatic transporters in the secretion of CEHCs from the liver into bile or plasma have yet to be elucidated.
In studies using insect microsomes expressing recombinant human CYP enzymes, Sontag and Parker showed that CYP4F2 metabolized γ-tocopherol, and to a much lesser extent α-tocopherol, to their respective 13′-OH-tocopherol metabolites (7
). In contrast we found that in rats given daily subcutaneous (SQ) α-tocopherol injections (10 mg /100 g body wt) to overload liver α-tocopherol capacity, hepatic CYP4F protein levels were unchanged. Surprisingly, there was a significant increase in metabolism of α-tocopherol, as evidenced by a 20-fold increase in hepatic 13′-OH-α-tocopherol levels (14
). Moreover, rat hepatic protein levels of CYP3A, 2B, and 2C family members increased (14
). These alterations in Phase I enzymes were not limited to massive α-tocopherol overloading; dietary α-tocopherol appears to be sufficient to alter xenobiotic metabolism. Specifically, hepatic Cyp3a protein levels were higher in mice fed an α-tocopherol sufficient (31 mg/kg diet) as compared to an α-tocopherol deficient (<2mg/kg diet) diet. Indeed, hepatic Cyp3a protein and α-tocopherol concentrations were correlated (16
To further examine the mechanisms of altered metabolism and disposition in response to α-tocopherol, we have studied rats given daily, SQ α-tocopherol injections (10 mg α-tocopherol/100 g body wt) for 18 days. Surprisingly, hepatic α-tocopherol and α-CEHC levels only increased up to day 9, then began to decrease (14
); hepatic protein levels of CYP3A, 2B and 2C increased concurrently with the increase in α-tocopherol and α-CEHC levels, while MDR1 protein increased concurrently with the subsequent decrease in hepatic levels of both α-tocopherol and α-CEHC (14
). These data indicated that mechanisms were in place to prevent the over-accumulation of α-tocopherol and that increasing α-tocopherol intakes, as well as administration of pharmacologic doses, modulate the expression of proteins involved in hepatic xenobiotic metabolism and excretion. However, the mechanism by which α-tocopherol might regulate these increases has not been investigated, nor has the extent to which α-tocopherol regulates all three phases of the hepatic xenobiotic pathways been investigated.
Alterations in hepatic xenobiotic pathways may occur at the gene level. Hepatic Cyp3a11 mRNA levels were elevated in mice fed 20 mg as compared with 2 mg α-tocopherol/kg diet; and were further increased in mice fed higher levels for a longer time (200 mg α-tocopherol/kg diet for 9 months) (17
). Expression of other xenobiotic pathway genes was not determined (17
Members of the CYP3A, 2B and 2C subfamilies are transcriptionally regulated by the nuclear receptors CAR (constitutive androstane receptor) and/or PXR (pregnane × receptor) (18
). In addition, PXR and CAR regulate MDR1 expression (20
). Nevertheless, in studies using HepG2 cells, Landes el al. (22
) showed that α-tocopherol was among the least effective of the vitamin E forms tested for the ability to activate PXR and another study using primary hepatocytes showed that only tocotrienols, not tocopherols, activated PXR (23
). Thus, the ability of α-tocopherol to alter xenobiotic pathways at the transcriptional level as a mechanism for increased hepatic CYP and MDR1 protein, with or without an involvement of nuclear receptors, as well as the number and extent to which additional genes involved in Phase I, II, and III are altered by increased hepatic α-tocopherol, requires further examination.
Based on the above observations, we hypothesized that α-tocopherol modulates hepatic xenobiotic pathways (Phase I, II, and III) by modulating gene expression and that determination of the specific subset of xenobiotic genes modulated by α-tocopherol would provide the necessary data for directing future experiments to investigate a role for nuclear receptors. To test this hypothesis we investigated the ability of elevated α-tocopherol intake to alter expression of genes involved in hepatic xenobiotic metabolism and excretion in mice using both whole genome microarray and real-time quantitative PCR (RT-qPCR) analyses.