The purpose of this study was to characterize the xenobiotic pathways involved in vitamin E metabolism based on the premise that α-tocopherol triggers modulation of CYP enzymes and xenobiotic transporters through PXR. Previously, we reported that CYP3A and MDR1 proteins were up-regulated in rats subcutaneously injected with vital E compared with those injected with saline [3
The findings concerning the effect of excess hepatic α-tocopherol on ABC transporters were consistent across all experiments. In our previous studies [3
] and the ones reported herein, ABCB1b
(MDR1 or p-glycoprotein), both the protein and the mRNA, increased with increased hepatic α-tocopherol. It, however, is not clear as to whether α-tocopherol, or its metabolite α-CEHC (or even γ-tocopherol, or γ-CEHC) were the stimulatory molecule(s) because all were well correlated with the gene changes observed ().
We also did a survey of various hepatic transporter genes and found that ABCG2
was up-regulated and OATP
was down-regulated by vitamin E injections. ABCG2 and ABCB1 proteins are both localized to the liver canaliculus, where they are thought to be involved in efflux of xenobiotics from the liver into the bile [36
]. ABCG2 expression is not limited to the liver, but “has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics” [37
]. Both ABCG2 and ABCB1 transport lipophillic molecules, but ABCG2 also has been reported to transport sulfates and glucuronides [36
], so may be important for trafficking CEHCs from the liver. In contrast, OATP is a transporter involved in influx to the liver from the blood stream [36
]. Thus, a decrease in OATP expression might limit the hepatic re-uptake of CEHCs. Further experimentation is needed to identify the roles of these transporters in regulating liver α-tocopherol concentrations. Moreover, the regulation of the transporters, themselves, is not well understood. PXR is not the only nuclear receptor implicated in regulation of ABC-transporters; other nuclear receptors relevant for their expression include the liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors α and γ [40
Although we confirm the up-regulation of ABCB1 protein and gene expression associated with increased hepatic α-tocopherol, we do not consistently find up-regulation of CYP3A protein or gene expression. It is generally accepted that PXR activates CYP3A gene expression [41
]. Given that α-tocopherol had been demonstrated to bind to PXR [10
] and that in mice dietary α-tocopherol could up-regulate mouse CYP3a11
], it seemed plausible that α-tocopherol would increase hepatic CYP3A in rats. However, our previous studies lacked an appropriate placebo; saline was used as the vehicle. For the studies described herein, we have obtained placebo solutions that contain all of the components, with the exception of vitamin E. When gene expression is compared between the placebo- and the saline only-injected animals only Sult2A1
expression were the same for saline and both placebos (); the two placebos had similar effects, except on CYP2B2
expression, which was less for the Vital E placebo. CYP3A2 protein was higher in the livers from rats injected with Vital E placebo compared with saline-injected, suggesting that there may be a component in the emulsifier that stimulates CYP3A2 protein expression.
The differences between the experiments previously published and the ones presented herein are difficult to reconcile. Despite our best efforts to mimic the previous conditions, there may be a component that was in the vital E solution that is no longer present. Other variables could include the amount of sodium pentobarbital administered, the degree of fasting, or there may be some additional differences in animals or diets, or some other unknown factors.
Both rat CYP3A1 and 3A2 are members of the CYP3A superfamily and are ~70% identical to human CYP3A4 [42
]. In the experiments presented herein, high liver α-tocopherol concentrations were associated with decreased CYP3A protein and mRNA expression; both CYP3A1 and 3A2 were tested to insure that we did not overlook their up-regulation. However, our findings are consistent with reports that α-tocopherol metabolism decreased PXR-mediated responses in mice [24
]. Moreover, in humans CYP3A does not appear to increase. The role of PXR in regulating CYP3A has led to the expectations that vitamin E supplements could dysregulate pharmaceutical drug metabolism [43
]. Three studies to test this hypothesis in humans have not
demonstrated decreased drug efficacy. When Leonard et al [45
] gave vitamin E supplements to hypercholesterolemic patients, the supplements did not alter efficacy of either simvastatin or lovastatin, two drugs metabolized via a CYP3A-dependent mechanism. Werba et al. [46
] approached this problem by studying the effect of simvastatin (20 mg/day) or pravastatin 40 (mg/day) on α- and γ-tocopherol concentrations in hypercholesterolemic humans. Although both drugs decreased circulating cholesterol and α-tocopherol concentrations, only simvastatin raised γ-tocopherol concentrations. These findings suggest that simvastatin and γ-tocopherol competed for the same sites for metabolism, while pravastatin, which is not dependent on CYP3A for metabolism, did not. Clarke et al [47
] hypothesized that α-tocopherol supplements would induce CYP3A4 in humans and thus decrease the plasma concentration of the CYP3A4 substrate, midazolam. Although they found increases in both plasma α-tocopherol concentrations and CEHC excretion, no changes in midazolam concentrations were detected. Taken together, these findings suggest that stimulation of PXR- or CYP3A-dependent metabolism tends to decrease, rather than increase, vitamin E metabolism. These outcomes are contrary to the expectation that vitamin E functioning as a PXR-ligand would increase PXR-responsive events. However, these reports are not consistent with the finding that α-tocopherol increased PXR-dependent increases in CYP3A and lead to increased vitamin E metabolism, as shown in a cell culture model using D-galactosamine-treated human hepatocytes [48
]. Thus, we are left with the quandary that there are no clear outcomes as to how and whether α-tocopherol alters CYP3A or PXR. Certainly, the findings from the experiments reported herein show that if PXR
gene expression is decreased CYP3A
expression is also decreased or unchanged (); however, these changes were not dependent on the liver α-tocopherol concentrations, suggesting that PXR was dependent upon some other factor.
Overview of results of vitamin E injection on gene expression studies relative to saline
We anticipated that rat hepatic SULT2A expression would also be PXR-dependent; however, this was not the case (). The transcriptional control of rodent SULT2A is not solely dependent upon regulation by PXR. Motifs required for transcriptional activation by PXR and other nuclear receptors, including constitutive androstane receptor, farnesoid X receptor and vitamin D receptor have been reported to be located in the 5′-flanking regions of rodent SULT2A genes [49
]. Thus, the lack of SULT2A regulation by α-tocopherol is not surprising, given the variety of possible stimulatory molecules.
In summary, our studies have demonstrated that an over-load of hepatic α-tocopherol increases its own metabolism, and increases transporters that are postulated to lead to increased excretion of both vitamin E and its metabolites. Based on the work of Parker’s group [7
] and the studies reported herein, it is clear that CYP4F2 is the only cytochrome P450 that initiates tocopherol metabolism. However, CYP4F2 also functions as a predominant leukotriene B4 and arachidonate omega-hydroxylase [52
], as well as being involved in vitamin K metabolism [53
]; thus is not specific only for vitamin E. Additionally, we have found that CYP4F does not change with hepatic α-tocopherol status [3
]. Thus, it would appear that vitamin E metabolism is largely dependent on the affinity of CYP4F2 for its substrates, as demonstrated by Sontag and Parker [8
]. The studies described herein emphasize that ABC-transporters, ABCB1 and ABCG2 are up-regulated in association with elevated hepatic α-tocopherol, suggesting they are involved in the disposition of either or both α-tocopherol and α-CEHC. Further studies examining the activity of these transporters are needed to confirm these observations.