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Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853
2To whom correspondence should be addressed. E-mail: ccm3{at}cornell.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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- and 
-tocopherol) are cytotoxic to some but not all cell types. Mouse macrophages were especially sensitive (40 µmol/L), whereas human hepatocytes and bovine endothelial cells were almost completely refractory (90 µmol/L). The fully methylated tocopherol, 
-tocopherol (-Toc), was not cytotoxic in any cell type tested. The cytotoxicity observed with -tocopherol (-Toc) was associated with 2 markers of apoptosis. Vitamer-specific cytotoxicity was not due to differences in cellular uptake/accumulation because both -Toc and -Toc accumulated equally in any cell type tested. In contrast, the cell-specific cytotoxicity was related in part to uptake/accumulation of the tocopherols. Macrophages accumulated nearly 5 times more tocopherol compared with hepatocytes cultured under similar conditions. To address the hypothesis that uptake accounted for the cell-specific sensitivity, we developed a macrophage "subtype" that was markedly resistant (>150 µmol/L) to -Toc. Under many different cell culture conditions (including human serum) uptake/accumulation of tocopherols was reduced in this subtype by 
50%. Further selection and evaluation of this phenotype, however, demonstrated no cytotoxicity even when cellular levels were elevated. Our results show that undermethylated tocopherols are cytotoxic to macrophages and that there are independent and selectable processes that determine cellular tocopherol uptake/accumulation and -Toc cytotoxicity.
KEY WORDS: • tocopherol • macrophage • cellular accumulation • vitamin E • toxicity
Tocopherols are a family of antioxidants that may be important in preventing chronic diseases (1–3). Although -tocopherol (-Toc)3 has been the most widely studied, other forms of tocopherol, particularly, -tocopherol (-Toc) and -tocopherol (-Toc), constitute nearly 60 and 20%, respectively, of tocopherols consumed in the United States (4). The greater emphasis on -Toc undoubtedly arises from the observation that -Toc and -Toc are only 10 and 1% as effective as -Toc, respectively, in experimental animal models of vitamin E deficiency. However, more recent studies emphasized the unique biological reactions of some non--tocopherols. For example, -Toc was shown to be a more effective inhibitor of peroxynitrite-induced lipid peroxidation in artificial liposomes (5), a characteristic correlated with greater reactivity toward electrophilic nitrogen oxides. In addition, -Toc was also recently shown to be more effective at inhibiting inflammatory mediator synthesis both in intact cells (6) and in vivo (7).
-Toc has received much less attention despite its considerable prominence as a dietary constituent. This is undoubtedly a consequence of its lower biological activity and the observation that both -Toc and -Toc levels in blood and tissues are only 0.20–0.01 that of -Toc (8). The remarkable difference between dietary intake (mentioned above) and subsequent tissue concentration of these tocopherols highlights a long-recognized metabolic discrimination between the vitamers. -Toc and -Toc are metabolized to water-soluble products (9) and/or excreted in bile (10) soon after ingestion; thus, they do not accumulate in blood or tissue. This "biodiscrimination" was described as a "salvage" pathway in which the metabolism and excretion of -Toc is prevented by a specific hepatic binding protein, -tocopherol transfer protein [see review (11)]. In addition, a cytochrome P450-mediated pathway of tocopherol metabolism that preferentially catabolizes -Toc over -Toc was recently described (12). It is not clear why these selective mechanisms evolved because untoward effects of naturally occurring tocopherols have not been reported. Some studies indicated that synthetic tocopherols, i.e., tocopherol-succinate, are capable of inducing cell death in vitro, but naturally occurring tocopherols do not appear to possess similar properties (13). Our present data show that -Toc and -Toc are cytotoxic in a cell-specific manner and that 2 selectable phenotypes exist in a mouse macrophage-like cell line that affect tocopherol accumulation and cytotoxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-Toc and -Toc) and Matreya (-Toc). All were natural RRR-tocopherols and were >98% pure as determined by GC-MS. A caspase-3 assay kit containing the colorimetric substrate, DVED-pNA, was obtained from Alexis Biochemicals. DMEM and other cell culture ingredients were purchased from Gibco. Fetal calf serum (FCS) was purchased from Hyclone. Protein dye was purchased from BioRad. DNA expression microarray chips (mouse 430A) were obtained from Affymetrix. Reagents for real-time PCR were obtained from Applied Biosystems. Delipidated FCS and other chemicals were purchased from Sigma Chemical.
Cell culture.
All cells were proliferating and maintained in DMEM supplemented with 2.2 g sodium bicarbonate/L and 10% FCS in an atmosphere of 5% CO2 and 95% humidified air at 37°C. Primary mouse macrophages were obtained from young adult male and female C57/Blk mice after the administration (i.p.) of 1 mL of 4% thioglycolate. Cells were obtained on d 3 after injection by peritoneal lavage as described previously (14). The NIH3T3 cell line was maintained in bovine calf serum (10%). Ethanolic solutions of tocopherols were added directly (dropwise) to serum while mixing and stored at 4°C for 24 h before the complete medium was prepared. In some experiments in which FCS was not employed, tocopherols were added to medium as a bovine serum albumin (BSA) (Ig-free) complex (tocopherol/BSA complex). This complex was prepared by adding 5 mL 1% Ig-free BSA to a dried film of tocopherol (5 mg) in a 25-mL Teflon-capped test tube. Samples were then mixed on a revolving wheel (12 revolutions/min) for 2–12 h at 4°C and filtered using 0.2-µm cellulose acetate filters. Tocopherol concentrations were determined by spectrophotometry and averaged 1.7 mmol/L over many preparations. This represented a yield of 70%. The suspension appeared homogenous and was stable for many weeks at 4°C with no visible separation or loss of potency. We analyzed this complex for the ratio of BSA to tocopherol after gel filtration (Sephadex, G100) chromatography. The molar ratio of BSA to tocopherol of void volume peak was 1:4000.
Cell cytotoxicity analysis. Stock cultures of cells (passages 4–15) were maintained and used during the exponential growth phase. Cells were subcultured into 24- (1.5 x 105 cells/well) or 48-well (7 x 104 cells/well) plates 24 h before experimental treatment. After treatment, the medium was replaced with warmed and equilibrated DMEM containing 0.05 g/L MTT (15). Cells were incubated for 30 min to 1.5 h, depending on cell type. Wells were washed once with an equal volume of 9 g/L saline. Then, 300 (48-well plates) or 400 µL (24-well plates) of dimethyl sulfoxide (DMSO) was added to each well and allowed to mix by agitation; 50 µL of glycine buffer (0.1 mol/L glycine, pH = 10) was then added to each well and mixed. We maintained conditions that provided a response between 0.3 and 1.2 OD units for the volume of DMSO in the respective plates. Under these conditions, a value in the linear response range was obtained. Protein concentration was determined using a protein dye (BioRad) and BSA as a standard.
DNA fragmentation and caspase-3 activity.
DNA fragmentation was determined by a procedure described by (16). This method isolates only fragmented DNA. Cells were treated with -Toc at 0, 30, or 60 µmol/L and at various times, and all cells were collected and lysed. Samples were then extracted and the fragmented DNA was treated with Rnase A and proteinase K. DNA fragments were then precipitated and separated by electrophoresis in 2% agarose gels. The resulting gel was stained with ethidium bromide. Caspase-3 activity was determined at various times after -Toc treatment (in complete medium) by colorimetric assay kits. Cells were treated with vehicle (0 µmol/L) or -Toc (60 µmol/L); 3,6, 9, 12, and 18 h later, the cells were washed and the cytosol prepared according to the manufacture’s instructions (Alexis Biochemicals). After protein determinations for each sample, equal amounts of cell cytosolic protein were added to buffer containing caspase-3 substrate (4 mmol/L), DVED-pNA. Optical density was determined at 400 nm.
Tocopherol cellular accumulation.
Twelve- (or 24-well) plates containing 3 x 105 (or 1.5 x 105) cells/well were seeded 24 h before uptake analysis. Hepatocyte (HepG2) cells were grown to confluence in 100-mm culture plates, resuspended in 10 mL of fresh medium, and 200 µL of resuspended cells were then seeded in 12-well plates and cultured for 3 d before uptake analysis. Accumulation was determined by the change in the cell-associated tocopherol concentration per milligram cell protein after a specified period of exposure to -Toc or -Toc. After exposure, adherent cells were washed once (for 15 min) with normal medium containing FCS (or 2% BSA in PBS) and then twice with an equal volume of PBS or DMEM. Cells were removed by scraping and then resuspended in PBS. Samples were sonicated for 15 s at 20% power (Sonics & Materials). Tocopherols were determined in cell sonicates by GC-MS as described previously (17). Protein concentrations were determined using a protein dye (BioRad) with BSA as a standard.
DNA array analysis.
Total RNA was prepared from each macrophage cell line (grown in duplicate 100-mm plates to 75% confluence) using the RNeasy Total RNA Isolation kit (Qiagen). Between 1 and 10 µg of total RNA from each sample was used to generate a high-fidelity cDNA, which was modified at the 3' end to contain an initiation site for T7 RNA polymerase. Upon completion of cDNA synthesis, 1 µg of product was used in an in vitro transcription reaction that contained biotinylated UTP and CTP, which are utilized for detection after hybridization to the oligonucleotide microarray. Full-length cRNA (20 µg), from both control and enriched samples, was fragmented in 200 mmol/L Tris-actetate (pH 8.1), 500 mmol/L KOAc and 150 mmol/L MgOAc at 40°C for 35 min. After fragmentation, all components generated throughout the processing procedure (cDNA, full-length cRNA, and fragmented cRNA) were analyzed by electrophoresis using the Agilent Bioanalyzer 2100 to assess the appropriate size distribution before microarray hybridization.
All samples were subjected to gene expression analysis via the Affymetrix Mouse 430A high-density oligonucleotide array, which currently queries 22,000 mouse probe sets. Currently, each gene on the array is represented by 11 pairs of 25-mer oligonucleotides that span the coding region for the 22,000 genes and expressed sequence tags represented (clear overlapping of genes is evident). Each probe pair consisted of a perfect match sequence that was complementary to the cRNA target and a miss match sequence that had a single base pair mutation in a region critical for target hybridization; this sequence served as a control for nonspecific hybridization. Hybridization, staining, and washing of all arrays was performed in the Affymetrix fluidics module according to the manufacturer’s protocol. Streptavidin phycoerythrin stain (Molecular Probes) was the fluorescent conjugate used to detect hybridized target sequences. The detection and quantitation of target hybridization were performed with a GeneArray Scanner 3000 set to scan each array twice at a factory-set photomultiplier tube level and resolution. All arrays referred to in this study were assessed for array performance before data analysis. Real-time PCR analysis was conducted for several genes (cathepsin H, ceruloplasmin, CD36 and 18s ribosomal RNA) on an ABI 7500 Real Time PCR System using reagents and protocols provided by Applied Biosystems (Assay-on-Demand). RNA was isolated using spin columns according to the manufacturer’s instructions (Ambion).
Data analysis. All data are presented as means ± SD. All experiments were repeated at least 3 times. When statistical analyses were required, data were analyzed by one-way ANOVA, and the means were compared using Dunnett’s procedure when comparing all means to a control or by multiple range test (Newman-Kuels) when comparing >2 means (18).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-Toc. Even though the concentrations of other vitamers only transiently approached these levels, our goal was to evaluate vitamer specificity; thus, we compared the various vitamers at equal concentrations. The results of representative experiments are shown (Fig. 1). Using complete medium (containing 10% FCS), cell viability was dramatically reduced (>90%) after 48 h of exposure to 60 µmol/L -Toc (Fig. 1A). The viability of cells exposed to -Toc under similar conditions was 50%, whereas -Toc had no effect. A relatively narrow range of concentrations was required for complete cytotoxicity (Fig. 1A, inset). Approximately 12 h of exposure to -Toc was sufficient to observe some change in cell viability. It is important to emphasize that cell protein was not altered at this time (data not shown), indicating no loss of cell number. Using DMEM containing delipidated FCS (10%) markedly enhanced cytotoxicity (leftward shift of the effective concentration) for both -Toc and -Toc but with little change in -Toc (Fig. 1C). These results most likely reflect the absence of lipoproteins and conditions that may promote the cellular accumulation of tocopherols.
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-Toc, most importantly primary mouse macrophages. Primary macrophages were equally sensitive to -Toc (Fig. 2A) relative to transformed mouse macrophages. Mouse fibroblasts and a human breast cancer cell line (MCF7) were also nearly as sensitive. On the other hand, endothelial cells and especially a hepatocyte cell line (HepG2) were almost completely refractory to 90 µmol/L -Toc (Fig. 2A).
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-Toc cytotoxicity is associated with apoptosis.
The synthetic forms of tocopherol were reported to induce apoptosis in some cell types (13). We therefore explored the possibility that -Toc might induce apoptosis in macrophages under our conditions. We employed 2 markers of apoptosis, DNA fragmentation and caspase-3 activity. After 12 h in the presence of 60 µmol/L -Toc (in complete medium), both DNA fragmentation and caspase-3 activity were increased (Fig. 3). We attempted to inhibit -Toc–induced cytotoxicity by the addition of up to 100 µmol/L of a caspase-3 inhibitor (Ac-DEVD-CMK, Calbiochem) but there was no evidence of protection (data not shown). This suggests that additional pathways of apoptosis may be involved in cell death.
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-Toc and -Toc.
We tested the hypothesis that -Toc preferentially accumulated relative to -Toc and that macrophages showed the greatest cellular accumulation of the 2 cell types. Short-term (6 h) cellular accumulation of both - and -Toc in both hepatocytes and macrophages was assessed in complete medium (containing 10% FCS). We found that hepatocytes accumulated 20% of both tocopherol vitamers compared with that observed in macrophages (Fig. 2B). These data strongly suggest that cellular accumulation of tocopherols, in general, may explain the differences among cell types. Moreover, they also indicate that the vitamer specificity is not the result of differences in cellular accumulation because neither hepatocytes nor macrophages exhibited a significant difference between vitamers (-Toc or -Toc). Subsequent experiments focused on the cell-type specificity and the cellular accumulation of tocopherols.
Selection of a cell subtype of macrophage that is resistant to -Toc cytotoxicity.
We addressed the possibility of selecting a "resistant" macrophage that possessed characteristics of tocopherol uptake/accumulation similar to those observed in hepatocytes. A "resistant" subtype macrophage cell line was obtained after 4 cycles of -Toc exposure (Fig. 4A). When activated with lipopolysaccharide and interferon-, these cells possessed a similar capacity (per mg protein) to synthesize nitric oxide. Additional selection was performed on this cell line (to be discussed later). We next addressed the question whether the selectable resistance to -Toc was associated with differences in the accumulation of tocopherols as previously observed in hepatocytes (as mentioned above). There are no reports of the cellular accumulation of -Toc in mouse macrophages. Both cell lines were evaluated under several different conditions, i.e., those of different durations and those in which tocopherol was "delivered" by both naturally occurring forms of tocopherol (human serum) and well as artificial preparations (FCS and tocopherol/BSA complex). The initial experiment observed uptake/accumulation over 6 and 24 h (Fig. 4B) in which tocopherols were present in complete medium (containing 10% FCS) as in the previous experiments. Over a 24-h period, the accumulation of both -Toc and -Toc was consistently lower for the "resistant" subtype especially at 24 h. The accumulation appeared not to change remarkably from 6 to 24 h of incubation. Again, there was little difference between the vitamers for either cell line, indicating that the cell accumulation of -Toc was similar to that observed with -Toc. We then focused on "initial uptake" in the absence of serum in the 2 cell lines. Short-term experiments evaluated accumulation/uptake within 1 h under serum-free conditions (Fig. 4C). Under these conditions, the "resistant" subtype accumulated 50% of both tocopherol vitamers compared with the parental line. A final experiment focused on the accumulation of a natural form of tocopherol occurring in human serum. Results from a representative experiment (Fig. 4D, left panel) or the mean (±SD) of 3 experiments (Fig. 4D, right panel) demonstrated that the accumulation of -Toc from human serum was 50% lower in the "resistant" subtype. Collectively, these data support the conclusion that the "resistant" subtype exhibited a lower uptake/accumulation of tocopherol under many different cell culture conditions.
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-Toc added to cultures of both cell lines; >90% of the tocopherol added to these cultures was recovered when both cells and medium were analyzed after 4 h of incubation (91.3 and 93% for the parental and resistant subtype, respectively). Collectively, these results suggest that transformation of tocopherols to known metabolites does not occur in these cells and therefore cannot explain the differences in tocopherol accumulation between the 2 cell lines.
Further selection of the "resistant" subtype.
The results presented above suggested that reduced cell accumulation of tocopherols might explain in part the resistance of the "resistant" subtype. It was apparent from further study, however, that at higher concentrations of -Toc, the "resistant" cell line experienced an 50% reduction in cell viability (Fig. 5A). We then initiated studies to "further select" the "resistant" subtype. The results (Fig. 5B) clearly demonstrated that at higher concentrations of -Toc, the original subtype (Subtype I) exhibited a loss in cell viability (of 50%) but the further selected cell line (Subtype II) was virtually insensitive (up to 160 µmol/L -Toc). A determination of the accumulation of both -Toc and -Toc in this further selected cell line demonstrated a more dramatic difference (now 70%) in the accumulation of either tocopherol (Fig. 5C). Thus, these data indicate that resistance to -Toc cytotoxicity follows (inversely) tocopherol accumulation. To address this directly, we focused on conditions that promoted increased accumulation of -Toc in the most resistant cell line (Subtype II). Under these conditions, we demonstrated that even when cell accumulation was comparable to that observed in the parental cell line, no cytotoxicity was observed (Fig. 6). These results strongly suggest that uptake/accumulation cannot be solely responsible for the resistance observed in the selected cell line. Overall, our results suggest that 2 phenotypes can be selected from the parental cell line, i.e., one that reflects processes controlling uptake/accumulation of tocopherols and another affecting -Toc cytotoxicity. Interestingly, the "resistant" subtype and the parental cell line are equally sensitive to staurosporine, a promiscuous protein kinase inhibitor, which is known to induce apoptosis in monocytes (19). This suggests that resistance to -Toc does not affect all paths to apoptosis.
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-Toc-"resistant" cell line (Subtype II). We evaluated the constitutive expression of nearly 22,000 genes and observed that the expressions of several genes were both remarkably higher and lower in the resistant subtype. Cathepsin H expression was increased nearly 200-fold, whereas carbohydrate sulfotransferase 1, sciellin, and ceruloplasmin increased from 70- to 17-fold (Table 1). The expression of 2 genes was reduced by 8-fold or more. These included CD36 and lipoprotein lipase. The relative expression of cathepsin H, ceruloplasmin, and CD36 in the parental and "resistant" subtype were verified by quantitative real-time PCR (data not shown). Whether these genes explain the phenotypes of the "resistant" macrophage cell line will be the focus of future research.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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- and -Toc were progressively more cytotoxic in macrophages but not in hepatocytes. Macrophages accumulated 5 times more tocopherol (both and ) compared with that observed in hepatocytes during a short-term exposure in cell culture, suggesting that cell type–specific cytotoxicity may correlate with cell uptake/accumulation. On the other hand, the accumulation of both - and -Toc was similar in each cell type (Fig. 2B), whereas only -Toc was cytotoxic. This observation strongly suggests that the differences in cytotoxicity among the various tocopherol vitamers (, , and ) cannot be explained on the basis of cell accumulation. This is contrast to the significant differences reported for these vitamers in preneoplastic and normal murine mammary epithelial cells (20,21). It is noteworthy that the vitamer-specific cytotoxicity appeared to correlate with the degree of methylation of the chroman ring. -Toc (fully methylated) was not cytotoxic whereas (lacking 1 methyl) and (lacking 2 methyls) were progressively more cytotoxic. We have also tested both ß- and 
-Toc, which are isomers of -Toc (i.e., lacking one methyl) and observed a similar cytotoxic profile (data not shown). Grau and Ortiz (22) indicated that the order of efficacy of tocopherols for phospholipase A2 inhibition in liposomes correlated with their biological activity and that the weaker inhibitors located progressively deeper into the bilayer. The "weaker inhibitors" were those with the greatest cytotoxicity (- and -Toc) in the present study. These observations suggest that the degree of methylation of the chroman ring maybe an important feature of cytotoxicity and that the observed differences could be related to the distribution of the tocopherols in the cell membrane.
The marked sensitivity of macrophages to -Toc was associated with the greater tendency of these cells to accumulate tocopherols (in general). This suggested that macrophages may be sensitive by virtue of their greater ability to uptake/accumulate tocopherols. Interestingly, various mammary epithelial cells (preneoplastic to malignant) showed a similar accumulation of -Toc but differing sensitivities (20). The interpretation in the present study was initially complicated by the numerous differences between macrophages and hepatocytes; there are many differences between these cell types that could potentially explain resistance. Subsequently, we selected a macrophage cell line for resistance to -Toc to explore the possibility that such a cell type might also exhibit lower tocopherol accumulation. We successfully selected a "resistant" macrophage subtype that was similar to hepatocytes in that they were completely resistant (up to 90 µmol/L) to normally lethal concentrations of tocopherols. More importantly, they appeared to accumulate less tocopherol under many different cell culture conditions including human serum (Fig. 4B–D). This condition is particularly unique and represents tocopherol delivered as a "naturally" occurring form found in human serum. The differences in tocopherol accumulation between the parental and the "resistant" macrophages were increased after further selection of the original "resistant" cell line. The extent of accumulation in the subsequent cell line (Subtype II) dropped to 20% of the parental (Fig. 5C). Collectively, these data suggested that selectable processes exist in macrophages that determine the extent of tocopherol cellular accumulation and potentially, the resistance to -Toc. The range of conditions employed in our studies was important because it illustrated that the mode of tocopherol "delivery" does not determine the differences observed among the cell lines. Ultimately, however, there was no evidence of cell death in this subtype (Subtype II) despite comparable cell accumulation (Fig. 6). This result suggested that factors other than uptake/accumulation must determine the resistance of the selected cell line.
To explore further the differences between the parental and "resistant" cell line that may explain the 2 phenotypes (tocopherol accumulation and -Toc resistance), we evaluated constitutive gene expression in both cell lines. Although the expression of many genes was altered (nearly 30 genes were increased by >4-fold), several were changed remarkably. Cathepsin H was increased nearly 200-fold in the "resistant" cell line whereas both CD36 and LPL were diminished >10-fold. All 3 genes may be involved in some aspect of tocopherol trafficking in macrophages. Cathepsin H is thought to be a marker of early endosomes (23). Endosomal transport may be a prominent mode of accumulation of tocopherols in macrophages. The class B scavenger receptor, CD36, has a remarkably diverse list of ligands including modified BSA and lipoproteins and a broad list of effects (24). It is interesting that -Toc was shown to reduce the expression of CD36 both in vivo (25) and in vitro (26,27). In addition, LPL was shown many years ago to be involved in the transfer of tocopherols from chylomicra and VLDL (28). It is not clear, however, that these latter components would be involved in uptake/accumulation of tocopherols when delivered as a BSA complex or in the absence of lipoproteins. Further work will be required to determine the role, if any, of these highly differentially expressed genes.
Finally, our results are consistent with the tendency of many organisms to preferentially retain tocopherol and to eliminate both and tocopherol. Despite comparable dietary intakes of these 3 vitamers, only -Toc accumulates to high levels in tissues (11). This biodiscrimination is not clearly understood especially because other forms of tocopherol (e.g., and ) have virtually the same protection against oxidative stress in vitro (29). Our results indicated that both - and -Toc are capable of causing cell death in macrophages. Although the concentrations of these tocopherols as employed in the current experiments may be observed only transiently (after a meal) in vivo, they are unlikely to be sustained in vivo due to the aforementioned biodiscrimination of tocopherols. Nonetheless, it is conceivable that cell functions could be altered at lower concentrations well before cell death is evident. Nevertheless, our data clearly indicate that the different tocopherol vitamers have very different biological activities toward macrophages and that our results are consistent with the general nature of tocopherol discrimination, i.e., retention of and elimination of both and .
In summary, our experiments demonstrated that some tocopherols are cytotoxic in certain cell types. Cytotoxicity was associated with methylation of the chroman ring in that the least methylated tocopherol () was most cytotoxic, whereas the fully methylated form () was not cytotoxic in any cell type. Macrophages were the most sensitive cell type tested compared with hepatocytes and endothelial cells. Cell-type sensitivity appeared to correlate with the ability of cells to accumulate tocopherols, in general. A "resistant" macrophage subtype was developed that was remarkably resistant to tocopherol. This phenotype was associated with a markedly diminished propensity of these cells to accumulate tocopherols (50–20% of parental). However, even under conditions in which cell accumulation was elevated, no cytotoxicity was observed. These data clearly demonstrate that processes other than uptake/accumulation must account for the remarkable resistance observed in the "resistant" subtype. Over all, our results are consistent with the "biodiscrimination" of tocopherols, i.e., preferential retention of -Toc and the elimination of other, potentially cytotoxic, forms of vitamin E.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: -Toc, -tocopherol; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; -Toc, -tocopherol; FCS, fetal calf serum; -Toc, -tocopherol.
Manuscript received 21 July 2004. Initial review completed 12 August 2004. Revision accepted 27 September 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Pryor, W. A. (2000) Vitamin E and heart disease: basic science to clinical intervention trials. Free Radic. Biol. Med. 28:141-164.[Medline]
2. Traber, M. G. (2001) Does vitamin E decrease heart attack risk? Summary and implications with respect to dietary recommendations. J. Nutr. 131:395S-397S.
3. Keaney, J. F., Jr, Simon, D. I. & Freedman, J. E. (1999) Vitamin E and vascular homeostasis: implications for atherosclerosis. FASEB J. 13:965-975.
4. Chow, C. K. (1985) Vitamin E and blood. World Rev. Nutr. Diet. 45:133-166 133–66.[Medline]
5. Christen, S., Woodall, A. A., Shigenaga, M. K., Southwell-Keely, P. T., Duncan, M. W. & Ames, B. N. (1997) -Tocopherol traps mutagenic electrophiles such as NOx and complements -tocopherol: physiological implications. Proc. Natl. Acad. Sci. U.S.A. 94:3217-3222.
6. Jiang, Q., Elson-Schwab, I., Courtemanche, C. & Ames, B. N. (2000) -Tocopherol and its major metabolite, in contrast to -tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 97:11494-11499.
7. Jiang, Q. & Ames, B. N. (2003) Gamma-tocopherol, but not alpha-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats. FASEB J. 17:816-822.
8. Traber, M. G. & Kayden, H. J. (1989) Preferential incorporation of alpha-tocopherol vs gamma-tocopherol in human lipoproteins. Am. J. Clin. Nutr. 49:517-526.
9. Swanson, J. E., Ben, R. N., Burton, G. W. & Parker, R. S. (1999) Urinary excretion of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans. J. Lipid. Res. 40:665-671.
10. Kayden, H. J. & Traber, M. G. (1993) Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J. Lipid. Res. 34:343-358.[Medline]
11. Brigelius-Flohe, R. & Traber, M. G. (1999) Vitamin E: function and metabolism. FASEB J. 13:1145-1155.
12. Sontag, T. J. & Parker, R. S. (2002) Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status. J. Biol. Chem. 277:25290-25296.
13. Yu, W., Simmons-Menchaca, M., Gapor, A., Sanders, B. G. & Kline, K. (1999) Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutr. Cancer 33:26-32.[Medline]
14. McCormick, C. C., Li, W. P. & Calero, M. (2000) Oxygen tension limits nitric oxide synthesis by activated macrophages. Biochem. J. 350:709-716.
15. van de Loosdrecht, A. A., Nennie, E., Ossenkoppele, G. J., Beelen, R.H.J. & Langenhuijsen, M.M.A.C. (1991) Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. J. Immunol. Methods 141:15-22.[Medline]
16. Herrmann, M., Lorenz, H. M., Voll, R., Grunke, M., Woith, W. & Kalden, J. R. (1994) A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res. 22:5506-5507.
17. Parker, R. S. & Swanson, J. E. (2000) A novel 5'-carboxychroman metabolite of gamma-tocopherol secreted by HepG2 cells and excreted in human urine. Biochem. Biophys. Res. Commun. 269:580-583.[Medline]
18. Steel, R. G. & Torrie, J. H. (1960) Principles and Procedures of Statistics 1960 McGraw-Hill New York, NY.
19. Schwartz, B. R., Karsan, A., Bombeli, T. & Harlan, J. M. (1999) A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells. J. Immunol. 162:4842-4848.
20. McIntyre, B. S., Briski, K. P., Gapor, A. & Sylvester, P. W. (2000) Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proc. Soc. Exp. Biol. Med. 224:292-301.
21. McIntyre, B. S., Briski, K. P., Tirmenstein, M. A., Fariss, M. W., Gapor, A. & Sylvester, P. W. (2000) Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells. Lipids 35:171-180.[Medline]
22. Grau, A. & Ortiz, A. (1998) Dissimilar protection of tocopherol isomers against membrane hydrolysis by phospholipase A2. Chem. Phys. Lipids 91:109-118.[Medline]
23. Claus, V., Jahraus, A., Tjelle, T., Berg, T., Kirschke, H., Faulstich, H. & Griffiths, G. (1998) Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes. J. Biol. Chem. 273:9842-9851.
24. Febbraio, M., Hajjar, D. P. & Silverstein, R. L. (2001) CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J. Clin. Investig. 108:785-791.[Medline]
25. Barella, L., Muller, P. Y., Schlachter, M., Hunziker, W., Stocklin, E., Spitzer, V., Meier, N., de Pascual-Teresa, S., Minihane, A. M. & Rimbach, G. (2004) Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats. Biochim. Biophys. Acta 1689:66-74.[Medline]
26. Devaraj, S., Hugou, I. & Jialal, I. (2001) Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages. J. Lipid. Res. 42:521-527.
27. Venugopal, S. K., Devaraj, S. & Jialal, I. (2004) RRR-alpha-tocopherol decreases the expression of the major scavenger receptor, CD36, in human macrophages via inhibition of tyrosine kinase (Tyk2). Atherosclerosis 175:213-220.[Medline]
28. Traber, M. G., Olivecrona, T. & Kayden, H. J. (1985) Bovine milk lipoprotein lipase transfers tocopherol to human fibroblasts during triglyceride hydrolysis in vitro. J. Clin. Investig. 75:1729-1734.
29. Saito, Y., Yoshida, Y., Akazawa, T., Takahashi, K. & Niki, E. (2003) Cell death caused by selenium deficiency and protective effect of antioxidants. J. Biol. Chem. 278:39428-39434.
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), human breast cancer cell line (MCF-7), mouse fibroblasts (NIH 3T3), human hepatocytes (HepG2), and bovine endothelial cells (Endo) after exposure to 
