Progress 10/01/00 to 09/30/04
Outputs
Over the life of this project, we have discovered that some forms of vitamin E are cytotoxic to certain cell types when cultured in vitro. The most important implications of our data are: 1) that prominent dietary forms of vitamin E possess a potential for untoward effects in certain cell types, and 2) that a rationale for the well-recognized biodiscrimination of different forms of vitamin E may have a basis in our results. It is well known that all mammals selectively retain and accumulate alpha-tocopherol despite a preponderance of other forms (i.e., gamma and delta-tocopherol) provided in the diet. The biological rationale for this mechanism has not been clarified despite the identification of the primary components involved (i.e., tocopherol-omega-hydroxylase and tocopherol transfer protein). Our observations suggests that the elimination of both gamma and delta tocopherol may have a basis in their potential cytotoxicity. Alpha-tocopherol possesses comparable
antioxidant activity but does not appear to possess similar cytotoxic potential. To evaluate the molecular mechanisms of cytotoxicity, we have selected a macrophage cell line (from the parental stock) that is resistant to delta-tocopherol cytotoxicity over a ten-fold higher concentration range. Cell accumulation in both cell lines was evaluated and found not to explain the resistance. Currently studies are ongoing to evaluate the molecular basis of this resistance. Constitutive gene array analysis has indicated some remarkable differences between the cell lines. Our working hypothesis is that some tocopherols (particularly delta and gamma) accumulate in certain membranes of the cell and, like free cholesterol, result a more ordered membrane structure, thus initiating events leading to apoptosis. One of the principal targets, we believe, is the endoplasmic reticulum, an organelle recently shown to be the target in free-cholesterol induced apoptosis. Overall, our data suggest that
tocopherol biodiscrimination may provide necessary protection against the accumulation of some forms of vitamin E. Currently, those forms of vitamin E constitute the major forms found in US diets.
Impacts
Our experimental data obtained using a cell culture model suggest that various isoforms of Vitamin E (i.e., those readily available in supplement form) may be deleterious to certain cells of the immune system. This has not been recognized before and may represent subtle effects that can only be demonstrated clearly in a simple cell culture system. In more complex systems, it may difficult to assess and as well may be tolerated over for considerable periods. Our goal is to explore the subtle effects (as observed in cell culture) in more complex organisms.
Publications
- McCormick, C.C., and R.S. Parker. 2004. The cytotoxicity of vitamin E is both vitamer- and cell-specific and involves a selectable trait. J Nutr, 134, 3335-42.
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Progress 01/01/03 to 12/31/03
Outputs
In a collaborative effort, we have been investigating a cell culture model of cellular effects of various forms of Vitamin E. We have found that certain forms of Vitamin E are remarkably cytototoxic to macrophages (but not other cell types) at physiologically relevant concentrations (less than 50 uM). Under all conditions studied, the rank order of cytotoxicity is delta greater gamma greater alpha tocopherol. This order corresponds to increasing methylation of the chromanol ring suggesting that methylation may be correlated with the observed effects. We have selected a macrophage cell line that is resistant (20 times) to normally toxic levels of delta tocopherol. Although tocopherol uptake is reduced in this cell line, this phenotype does not explain the resistance. Our most recent observations indicate that tocotrienols (forms of vitamin E with unsaturated phytyl tails) are especially cytotoxic even in the resistant cell line. Both the parental cell line and the
resistant cell line are equally sensitive to tocotrienols. We concluded that these forms of vitamin E may interact with different intercellular targets when compared to the tocopherols. The mechanism of cell death induced by tocopherols may be related to a disruption of cellular free cholesterol. Very recent observations indicate that phosphatidylcholine synthesis is increased markedly (4-fold) by exposure to vitamin E, a characteristic that is similar to that seen with elevated free cholesterol. In fact, changes in free cholesterol have been reported recently to explain cell death of macrophages in atherosclerotic lesions leading ultimately to infarction. Our data suggest that tocotrienol supplementation may have unrecognized untoward effects which may oppose the purported antioxidant benefits. Our future research will focus on the hypothesis that vitamin E mimics free cholesterol and thereby initiates molecular events of cell death via apoptosis. These events have recently been
described to include activation of the unfolded protein response in the endoplasmic reticulum. We will evaluate similar processes in macrophages during exposure to various forms of vitamin E and in more complex organisms exposed to high dietary Vitamin E.
Impacts
Our experimental data obtained using a cell culture model suggest that various isoforms of Vitamin E (i.e., those readily available in supplement form) may be deleterious to certain cells of the immune system. This has not been recognized before and may represent subtle effects that can only be demonstrated clearly in a simple cell culture system. In more complex systems, it may difficult to assess and as well may be tolerated over for considerable periods. Our goal is to explore the subtle effects (as observed in cell culture) in more complex organisms.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Vitamin E is an important nutrient that is thought to be critical in our defense against chronic diseases such as heart disease and certain cancers. Currently, most of the Vitamin E found in diets common in the US is derived from two sources of dietary oil, soybean and corn. These oils are unusually rich the forms of two forms of Vitamin E (gamma and delta tocopherol) that possess lower biological activity. Alpha tocopherol is the third and most abundant vitamer that is the form of Vitamin E found in supplements. While conducting unrelated research, our laboratories have recently discovered that both gamma and especially delta tocopherol are capable of killing some cells in culture at concentrations that are biologically relevant. Alpha tocopherol, in contrast, was not cytotoxic under these conditions. We have begun to explore the biological basis of the cell killing observed under our conditions. Some cell-types are completely resistant while others, most notably
macrophages, are very sensitive. In the past year, we have determined that naturally occurring forms of tocopherol, i.e., those bound to lipoproteins, are capable of cell killing. Delta tocopherol at concentrations as low as 8 uM was cytotoxic in the presence of human acetylated LDL. Most importantly, this cytotoxicity could be completely prevented by a reagent (dextran sulfate) that blocks acetylated-LDL uptake by scavenger receptors. Thus we have shown that tocopherol when delivered in a biologically relevant form is capable of initiating cell death. We also have demonstrated that normal lipoproteins both LDL and HDL are protective. We attribute this protection to the ability of these particles to sequester tocopherols. Interestingly, HDL was 2 times more effective on the basis of cholesterol content. Finally, we have recently selected a resistant subtype of macrophage from the original parental line and are beginning to characterize this resistant cell type. Our preliminary studies
suggest that these cells accumulate tocopherols at lower rates when compared to parental cells. This phenotype is characteristic of other resistant cells, e.g. hepatocytes.
Impacts
This research will reveal a biological activity of an important nutrient that has not be characterized previously. Our research has the potential to change our dietary recommendations regarding food sources of Vitamin E.
Publications
- McCormick, C.C. 2002. Diffusion does not play a major role in the absorption of dietary calcium in normal adults. J. Nutrition 132:3428-3430.
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Progress 01/01/01 to 12/31/01
Outputs
Processed foods constitute a major source of dietary tocopherols in the US. The two most important contributors, corn and soy oils, contain forms of vitamin E (gamma and delta tocopherol) which are not found in supplements (alpha tocopherol). Recent work has suggested that gamma (g) tocopherol may have unique biological properties not shared by a-tocopherol. Our research represents a collaborative effort focused on unique properties of gamma and delta (d) tocopherol. Our research has demonstrated, for the first time, that g-tocopherol and especially d-tocopherol can induce cell death in some cells at physiologically relevant concentrations. Of the cell types tested, macrophages were the most sensitive while hepatocytes were completely refractory. Cell culture conditions profoundly influenced the concentration at which cell death occurred - removal of lipoproteins markedly enhanced cytotoxicity. Hepatocytes accumulate much less tocopherol when compared to macrophages.
Although there was little difference in the accumulation (at >6hr) of either a-tocopherol or d-tocopherol in both cell types, only d-tocopherol induced cell death. Using the macrophage cell line, we attempted to "select" for d-tocopherol sensitivity. Macrophages were selected for their ability to tolerate high concentrations of d-tocopherol. A cell-line was developed that was completely refractory to normally lethal levels of d-tocopherol. Accumulation of tocopherol by these cells was found to be lower when compared to that observed in parental macrophages. These data suggest that accumulation of tocopherols by cells may be governed by a selectable trait. Overall, our results show that: 1) both g- and d-tocopherol are potentially cytotoxic at physiologically relevant concentrations in some cell types, 2) a-tocopherol accumulates similarly in sensitive cells but is not cytotoxic and 3) cellular accumulation of tocopherols is determined by a selectable trait. Our data imply that the
mechanisms of tocopherol discrimination in vivo, in which alpha tocopherol is selectively retained and other forms eliminated, may have evolved to prevent the accumulation of cytotoxic forms of this vitamin. It is conceivable that conditions exist in vivo where this mechanism is either diminished or defective and that under these conditions dietary sources of other tocopherols become critical. Currently, the vitamers that provide the majority of dietary tocopherols in the U.S. are g-tocopherol and d-tocopherol, forms shown to be potentially cytotoxic.
Impacts
Our research has revealed novel biological properties of vitamin E. Most importantly, our results indicate that some forms of vitamin E may be cytotoxic to some cell types. Since humans possess biological mechanisms to selectivity eliminate the cytotoxic forms, our data implicate a novel rationale for these processes.
Publications
- McCormick, C. and R.S. Parker. 2001. Cytotoxicity of Tocopherols: A cell specific and selectable mechanism involving cellular uptake. FASEB J. 15:55 (abstract #LB298)
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