In the current study we have used various doses of α-tocopherol injected SQ in rats to increase plasma and tissue α-tocopherol levels and then determined α-CEHC, CYP and MDR1 expression in selected tissues. Importantly, our data shows that mechanisms are in place to prevent the excess accumulation of both α-tocopherol and α-CEHC in extrahepatic tissues.
In rats given daily SQ α-tocopherol injections (10 mg/100 g body wt) for 18 days, hepatic α-tocopherol and α-CEHC levels increased only up to about day 9, then began to decrease [4
]. Interestingly, hepatic CYP3A almost immediately doubled, while the MDR1 proteins increased concurrently with decreasing α-tocopherol concentrations [4
]. These results indicated that mechanisms that prevent the over-accumulation of α-tocopherol in liver are up-regulated in rats given pharmacologic doses of α-tocopherol and that administration of pharmacologic doses of α-tocopherol modulates hepatic expression of proteins involved in xenobiotic metabolism and excretion. Thus, we were interested to determine the extent to which extrahepatic tissues were protected from over-accumulation of α-tocopherol and to further elucidate the ability of α-tocopherol to modulate their xenobiotic metabolism and excretion pathways.
Interestingly, in contrast to hepatic α-tocopherol concentrations that increased 15-fold in rats given the highest α-tocopherol dose, only the α-tocopherol concentrations in lung, muscle, heart, jejunum and duodenum increased more than 2-fold, with the duodenum having the highest fold change (3.2-fold). The increase in the duodenum may be due in part to enterohepatic circulation of α-tocopherol excreted from the liver into the bile [24
]. The 2-fold increase in plasma α-tocopherol concentration is consistent with studies where humans were given either 400 or 800 IU RRR
-α-tocopherol/day for 8 weeks and plasma α-tocopherol concentrations were found to increase approximately 2-fold at both dose levels [26
]. These data indicate that the body has the ability to eliminate “excess” α-tocopherol, even when pharmacologic doses are administered. Moreover, plasma and extrahepatic tissue α-tocopherol levels did not increase more than approximately 3-fold. The exception was the liver where excess α-tocopherol appears to be sequestered and then rapidly metabolized and/or excreted.
Systemic regulation of α-CEHC levels has not to our knowledge been previously reported. Our data shows that α-CEHC increased in liver, lung and kidney with increasing dose of α-tocopherol. However, even at the highest administered α-tocopherol dose, kidney α-CEHC levels were one-third and lung α-CEHC levels were one-fortieth of liver α-CEHC levels. In addition, α-CEHC levels in both lung and kidney began to plateau following administration of greater than 2 mg α-tocopherol/100 g body wt. These data indicate that, like α-tocopherol, mechanisms are in place to prevent the accumulation of α-CEHC in extrahepatic tissues.
Like CYP4F2 in humans, CYP4F1 is the most abundant CYP4F isoform found in the rat liver (80%) and kidney (95%), and has been shown to have similar substrate specificity to human CYP4F2, i.e., ω-hydroxylation of arachidonic acid and leukotriene B4
]. CYP4F2 has been identified by in vitro studies as the putative tocopherol hydroxylase [3
], and as such may be expected to increase under conditions of increased α-tocopherol metabolism. However, in our previous study hepatic α-CEHC concentrations increased more than 80-fold, while hepatic CYP4F protein expression remained unchanged [4
]. In the current study, kidney CYP4F1 levels were unchanged and lung CYP4F1 remained undetectable despite significantly increased α-CEHC concentrations in both tissues. Thus, our data indicate that SQ administration of pharmacologic doses of α-tocopherol does not modulate CYP4F protein levels in rat liver, lung or kidney. Furthermore, CYP3A protein was not detected in kidney and was only expressed at very low levels in lung. Together, these data suggest the modest increase in lung α-CEHC levels is likely a result of plasma delivery and not metabolism of α-tocopherol in situ. Presumably, the increase in kidney α-CEHC levels is due to its excretory function [2
]. However, since CYP4F1 is constitutively expressed in the kidney, α-tocopherol metabolism within the kidney may be an additional source of kidney α-CEHC.
MDR1 and CYP3A overlap with respect to substrate specificity and coordinate modulation of MDR1 and CYP3A protein expression in the liver has been demonstrated for several xenobiotic compounds [13
]. In contrast to the numerous studies investigating the regulation of hepatic MDR1, regulation of lung MDR1 has received very little attention and, in a search of the literature, we were unable to find information with respect to coordinate regulation of MDR1 and CYP3A proteins in the lung. Interestingly, several agents, including clotrimazole (CLOT) and a mixture of DEX + pregnenolone 16β-carbonitrile (PCN) in rats, and rifampicin exposure in rabbits [37
], which increase hepatic CYP3A protein have failed to induce lung CYP3A protein or activity levels. Unfortunately, MDR1 protein expression in either the lung or liver was not studied [37
Recently, we demonstrated in rats that supplementation with 10 mg α-tocopherol/100 g body wt increases the expression of hepatic MDR1 and CYP3A proteins [4
]. To further elucidate the ability of α-tocopherol to modulate xenobiotic pathways in rat lung, we determined the ability of various α-tocopherol doses given daily by SQ injection to coordinately modulate CYP3A and MDR1. Importantly, lung MDR1 protein levels increased with increasing α-tocopherol concentrations, while lung CYP3A protein expression was unaltered. These data suggest that the mechanism(s) by which α-tocopherol increases CYP3A expression is: 1) tissue specific and 2) independent of the mechanism by which α-tocopherol increases lung MDR1 protein expression.
In addition to CYP3A, we determined the ability of α-tocopherol to alter the expression of CYP1A protein in the lung and liver of rats. The CYP1A subfamily are key enzymes in the metabolic activation of numerous environmental xenobiotics, including many that enter the body via the respiratory system. Metabolic activation by the CYP1A subfamily of enzymes results in the production of highly reactive intermediates that have been shown to damage DNA [39
]. Thus induction of this enzyme in either the liver or lung may lead to increased tissue damage. Importantly, CYP1A protein was not changed by pharmacologic doses of α-tocopherol.
Hepatic expression of members of the CYP3A subfamily, as well as that of the MDR1 transporter, is regulated by nuclear receptors [15
]. Specifically, both CAR (constitutive androstane receptor) and PXR (pregnane × receptor) have been shown to regulate CYP3A; PXR has been shown to regulate MDR1 expression [44
]. Landes et al. [46
] demonstrated that various forms of vitamin E activated PXR expressed in HepG2 cells. However, α-tocopherol was one of the least effective of the forms tested. Conversely, another study showed that only tocotrienols, and not tocopherols, activated PXR in both intestinal LS180 cells and primary human hepatocytes [47
]. Chirulli et al. [37
] have demonstrated a low level of constitutive expression of CAR mRNA, but no expression of PXR mRNA, in rat lung. These data indicate that mechanisms of xenobiotic pathway induction are tissue specific and suggest it is unlikely that α-tocopherol is modulating lung MDR1 via the PXR receptor. Therefore, further studies in vivo are needed to determine the mechanism(s) by which α-tocopherol modulates lung MDR1 expression, as well as the mechanism(s) for modulation of hepatic CYP and MDR1 expression.
In conclusion, α-tocopherol concentrations increased 2-fold or less in most extrahepatic tissues and did not exceed 3.5-fold in any tissue, except the liver, even at the highest levels of α-tocopherol administered. In addition, our results indicate that unlike hepatic CYP3A expression, lung and kidney CYP3A expression is not altered by excess α-tocopherol. Furthermore, no α-tocopherol dose altered either hepatic or extrahepatic expression of either CYP1A or CYP4F proteins. Thus, induction of CYP enzymes by α-tocopherol is both tissue and CYP-subfamily specific. Importantly, lung MDR1 expression increased with α-tocopherol concentrations. Because MDR1 is responsible for the elimination of numerous toxic xenobiotics and based on its location in the alveolar membranes of both the rat and human respiratory system, we propose that induction of this transporter in the lung may represent an important mechanism for protection of the lung from exposure to xenobiotics and their metabolites, as well as a mechanism for delivery of α-tocopherol to the alveolar space.