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Nutrient Metabolism

Long-Chain Carboxychromanols Are the Major Metabolites of Tocopherols and Tocotrienols in A549 Lung Epithelial Cells but Not HepG2 Cells^1,2

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853

³To whom correspondence should be addressed. E-mail: rsp3{at}cornell.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Human lung type II cell derived A549 epithelial cancer cells and HepG2 hepatocytes constitutively express cytochrome P4504F2, a P450 we previously identified as a tocopherol--hydroxylase. To determine if A549 cells would metabolize tocochromanols via the -hydroxylase pathway, we compared the metabolism of tocopherols (-, -, -TOH) and tocotrienols (-, -, -T3) in these 2 cell lines. Cultures were incubated with -, -, or -TOH, or the analogous T3s, and synthesis of their metabolites quantitated by GC-MS. A549 cells metabolized all tocochromanols 2–3 times more extensively than HepG2 cells (P < 0.001) except -TOH, a difference not related to cell uptake of substrate but rather was reflective of greater microsomal TOH--hydroxylase enzyme activity. Notably, 9'-carboxychromanols were the major metabolites of all - and -TOHs and T3s in A549 cultures, whereas 3'- and 5'-carboxychromanols predominated in HepG2 cultures. Accumulation of 9'-carboxychromanols in A549 cultures was due to their inefficient conversion to 7'-carboxychromanols relative to HepG2 cells. Sesamin inhibited tocochromanol metabolism in both cells types, and neither cell type exhibited evidence of alternative (sesamin-insensitive) pathways of metabolism. TOH--hydroxylase activity was undetectable in rat primary lung type II cells, suggesting that expression of activity was associated with transformation of normal type II cells to cancer cells. Long-chain carboxychromanol metabolites of -TOH and other forms of vitamin E can be biosynthesized in A549 cultures for assessment of their biological activity, including their potential inhibition of synthesis of inflammatory mediators.

KEY WORDS: • tocopherols • tocotrienols • metabolism • -oxidation • A549 • HepG2

We previously reported that tocopherols (TOH)⁴ are catabolized to water-soluble metabolites by a cytochrome P450 (CYP)-mediated process initiated by -hydroxylation of a terminal methyl group of the phytyl tail by CYP4F2 (1), an enzyme first characterized as a leukotriene B4 -hydroxylase (2). The initial hydroxylation is followed by a dehydrogenation yielding the 13'-carboxychromanol, and subsequent truncation of the phytyl side chain by sequential removal of 2 or 3 carbon moieties. All expected intermediates in the side-chain truncation pathway were previously characterized by GC-MS (1). Figure 1 shows the structure of -T3 and its 9' and 3' carboxychromanol metabolites. The ultimate products of this pathway of tocochromanol catabolism are the ß-carboxyethyl-6-hydroxychromans (CEHC or 3'-carboxychromanols), which are excreted in urine. This pathway metabolized -TOH much more extensively than -TOH, leading us to propose its role in the preferential retention by humans of -TOH relative to other tocochromanols. Yamane and Abe (3) reported that A549 lung epithelial cells and HepG2 hepatocytes, both human cell lines, expressed CYP4F2 mRNA. Additionally, A549 cells exhibited 2 times greater CYP4F2 activity compared with HepG2 cells when assessed by leukotriene B4 -hydroxylase activity. A549 cells are the current model of choice for human type II pneumocytes. The A549 cell line was originally derived from a human lung carcinoma of type II origin and expresses many functional characteristics of primary type II cells (4). Based on this evidence, we hypothesized that A549 cells would exhibit TOH--hydroxylase activity and that the activity would exceed that of HepG2 cultures and be present in rat lung primary type II pneumocytes.

View larger version (27K): [in this window] [in a new window]   FIGURE 1 Structure of -T3, indicating carbon numbers of the carboxy or hydroxy metabolites of this and other tocochromanol substrates. Also shown are the structures of 9'--carboxy(3'-en)chromanol, the major metabolite of -T3 in A549 cultures, and 5'--carboxychromanol, the major metabolite of -T3 in HepG2 cultures.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

    Cell culture. A549 (CCL-185) cells, HepG2 cells (subtype C3A, CRL-10741), and H69AR (CRL-11351) cells were obtained from American Type Culture Collection. Cells were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum (FBS; Cellgro by Mediatech) without antibiotics. For comparison of TOH and T3 metabolism, cells were used 2 d after reaching confluency. Vitamin E compounds (concentrated stocks in ethanol) were added to FBS and allowed to stand overnight prior to dilution with Dulbecco’s Modified Eagle Medium and addition to cell cultures. Sesamin or ketoconazole were dissolved in ethanol and added to cultures 4 h prior to, and again after, addition of tocochromanol-supplemented media. All results shown are representative of multiple independent experiments.

To measure the disappearance of tocochromanols from cells, cells were first incubated with growth medium containing a combination of 10 µmol/L d₆--TOH, 15 µmol/L -TOH, and 18 µmol/L -T3. This combination was determined in preliminary experiments to result in similar intracellular concentrations of the 3 vitamers within 48 h of culture, given their observed differential rates of catabolism (see below). Deuterium-labeled -TOH was used to permit differentiation from the small amount of endogenous (FBS-derived) -TOH in the cultures. Tocochromanol-enriched cultures were washed twice with PBS, then incubated in growth media containing no added vitamin E for 0, 24, and 48 h, after which the cells were washed and analyzed for tocochromanol content. For the 48-h cultures, the media was changed after 24 h, and media tocochromanols from the first and second 24-h periods were summed to obtain the total mass of nonmetabolized tocochromanol secreted into the media. Cell protein was measured by the Bio-Rad (Bio-Rad Laboratories) method, and cell unesterified cholesterol was measured by GC-MS as described below for TOH, with the appropriate detector response factor. Cell unesterified cholesterol and protein were found to be equally reflective of cell mass.

    Analysis of TOH, T3s, and their metabolites. Metabolites were quantitated in media (only trace amounts were associated with cells) and tocochromanols (substrates) were quantitated in both media and cells, depending on the experiment. To measure cell-associated TOHs and T3s, cultures were washed twice with cold PBS and detached with a spatula. Tocochromanols were extracted from cell suspensions or media by addition of 2 volumes of absolute ethanol and 5 volumes of 90:10 hexane:methyltertbutylether, using d₉--TOH as an internal standard. For analysis of metabolites, media samples were acidified to pH 1.5 with hydrochloric acid (3 mol/L) and extracted with 3 volumes ethyl acetate after addition of d₉-3'--carboxychromanol internal standard. Cell and media extract residues were silylated by redissolving in 50 µL pyridine (Pierce Chemical) and 50 µL N,O-bis[trimethylsilyl]trifluoroacetimide (Pierce Chemical), and heating at 60°C under nitrogen atmosphere for 30 min. Trimethylsilyl derivatives of tocochromanols and their metabolites were quantified by GC-MS as previously detailed (1). Briefly, TOHs and T3s were resolved isothermally at 280°C using an HP-1 capillary column (Agilent Technologies) and helium carrier, and metabolites by temperature gradient. Detection was by selected ion monitoring. TOHs and T3s were quantitated against the d₉--TOH internal standard, adjusting for predetermined detector response differences. Metabolites were quantitiated against the d₉--CEHC standard.

    TOH--hydroyxlase activity of cell-free homogenates of HepG2 and A549 cells. Cell-free homogenates were prepared from HepG2 and A549 plates that had reached confluency in 100-mm culture plates. The cells were scraped into 100 mmol/L KH₂PO₄ buffer (pH 7.4) and gently centrifuged to sediment whole cells. The supernatant was removed, and the cells were resuspended in buffer for homogenization. Because a Polytron-homogenizer was not sufficient to break the cells, a motorized Potter-Elvehjem Teflon homogenizer was subsequently used. Postnuclear homogenates were prepared in a similar manner for each cell type, except the cell homogenate was additionally centrifuged at 2000 x g to clear the nucleus and cell-membrane fragments. The supernatant was subsequently centrifuged at 100,000 x g to collect the remaining subcellular fractions, which were resuspended in KH₂PO₄ buffer. Total protein was measured for each homogenate using the BioRad method. TOH--hydroxylase activity was measured for each fraction with 25 µmol/L tocochromanol-BSA complex described previously (1) preincubated for 1 h at 37°C with either 400 mg/L cell-free homogenate protein or 200 mg/L postnuclear homogenate. The reaction was initiated with the addition of 0.5 mmol/L NAD(P)H and NAD for 20 min, and stopped with the addition of 0.1 volume of 3 mol/L HCl. Tocochromanols and metabolites were extracted, derivatized, and analyzed as previously detailed (1). The end point of this assay is the sum of the 13'-OH and 13'-COOH metabolites, because no shorter-chain metabolites are formed under these conditions (1).

    Assay of TOH--hydroxylase activity in rat primary type II alveolar pneumocytes. Primary type II pneumocytes were isolated from rat lung as previously described (5). Isolated cells, in 35-mm culture dishes, were incubated in Eagles Minimal Essential Medium containing 10% FBS previously enriched with either -TOH or -TOH. Final substrate concentrations in the media were 30 µmol/L -TOH and 30 or 45 µmol/L -TOH. Cultures were incubated for 24 h, after which media was collected for analysis of -oxidation products by GC-MS as described above.

    Statistical analysis. Differences in metabolism of TOHs and T3s by A549 and HepG2 cell cultures or cell-free homogenates were examined by 2-way analysis of variance using general linear models, followed by multiple comparisons applying Bonferroni correction. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Comparative metabolism of tocochromanol by A549 and HepG2 cultures. GC-MS analysis of media from A549 cultures incubated with TOHs revealed the presence of the full range of carboxychromanol metabolites we previously reported to occur in HepG2 cultures. These included the 3'-, 5'-, 7'-, 9'-, 11'-, and 13'-carboxychromanol metabolites (Fig. 2). In addition, the immediate TOH--hydroxylase product, 13'-hydroxychromanol, was observed in - and -TOH cultures but that of -TOH cultures was below the limit of detection. The analogous tail-truncated metabolites of the T3s were biosynthesized, although some contained fewer double bonds than expected, as first reported by Birringer et al. (6) in HepG2 cultures incubated with - or -T3. These included the 5'-carboxychromanol with no double bond, the 7'- and 9'-carboxychromanols with one double bond, and the 11'-carboxychromanol with 2 double bonds. In addition, we observed the presence of the 13'-carboxychromanol, containing 2 double bonds (vs. 3 expected), and the 13'-hydroxychromanol, containing 3 double bonds, for all 3 T3 substrates.

View larger version (19K): [in this window] [in a new window]   FIGURE 2 Comparative distribution of individual tocochromanol metabolites produced by A549 and HepG2 cultures. A549 or HepG2 cultures were incubated with 25 µmol/L -TOH (A) or -T3 (B) for 48 h. Metabolites included the 3', 5', 7', 9', 11', and 13' carboxychromanols, and the 13'-hydroxychromanols. Data are means and SD from triplicate cultures per treatment. tr, trace. Similar results were obtained using 25 µmol/L -TOH and -T3 as substrates.

HepG2 and A549 cultures exhibited large differences in the overall extent of metabolism of substrate TOHs and T3s. A comparison of total metabolite formation over 48 h of incubation by the 2 cell types for 6 TOHs and T3s is presented in Figure 3. A549 cultures metabolized all substrates (except -TOH) significantly more than HepG2 cultures (P < 0.001). Among the various substrates, T3s were metabolized to significantly greater extents than their corresponding TOHs (P < 0.001), and vitamers were metabolized to significantly lesser extent than and vitamers (P < 0.001). The higher rates of metabolism by A549 cells could not be accounted for by a greater substrate uptake, because HepG2 cells exhibited similar or higher cell-associated substrate levels than A549 cells at the end of the incubation period (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 3 Comparison of tocochromanol metabolism by A549 and HepG2 cultures. A549 and HepG2 cultures were incubated with various individual tocochromanol substrates [25 µmol/L (62.5 nmol per 2.5 mL culture) for 48 h. The mean amounts of protein were 0.59 mg/plate]. The sum of concentrations of all detected metabolites was expressed per mg cell protein. Data are means and SD from triplicate cultures per treatment; *denotes a significant difference between cell types for the indicated substrate (P < 0.001).

To rule out the possibility that the vitamer difference in metabolism of tocochromanols was related to differential substrate uptake or secretion during continuous culture with substrate, A549 cells were loaded simultaneously to similar levels with d₆--TOH, -TOH, and -T3, then incubated in nonsupplemented media. A comparison of the rate of disappearance of the 3 substrate tocochromanols from the cells is presented in Figure 4. By 48 h, only trace quantities of -T3 remained in the cells. Approximately 10% of the original -TOH remained associated with the cells at 48 h, whereas 67% of starting d₆--TOH remained. Only 6–7% of the initial cell-associated mass of the 3 vitamers was recovered in the media over the 48-h period, with the remainder recovered as metabolites. There was no evidence of conjugated metabolites based on a lack of effect of ß-glucuronidase or sulfatase treatment of media prior to extraction.

View larger version (15K): [in this window] [in a new window]   FIGURE 4 Comparison of elimination of cell-associated d6--TOH, -TOH, and -T3 from A549 cultures. Cultures were incubated for 48 h with growth media supplemented with a combination of the 3 tocochromanols at concentrations previously determined to yield similar intracellular concentrations of these 3 tocochromanols. Cultures were then washed and incubated for the indicated times in nonsupplemented growth media. Data are means and SD of triplicate cultures at each time point.

View larger version (22K): [in this window] [in a new window]   FIGURE 5 Comparative inhibition -T3 metabolism by sesamin and ketoconazole in A549 cultures. Cultures were incubated in the presence of varying concentrations of sesamin or ketoconazole. Metabolites were quantified in the culture media, and the sum of concentration of all metabolites plotted as a function of inhibitor concentration. Data are means and SD from triplicate cultures per treatment.

View larger version (31K): [in this window] [in a new window]   FIGURE 6 Effect of sesamin on -T3 metabolite distribution and on recovery of substrate tocochromanols in A549 cultures. A: A549 cultures were incubated with 25 µmol/L -T3, with or without 5 µmol/L sesamin for 48 h. Metabolite notations are similar to those of Figure 2. B: A549 cultures were incubated with -TOH or -T3 (25 µmol/L, 62.5 nmol per plate), with or without 5 µmol/L sesamin, for 48 h. Metabolites in the culture medium and remaining nonmetabolized substrates in cells and medium were quantified by GC-MS. Data are means and SD from triplicate cultures per treatment. tr, trace.

To examine whether alternative pathways of tocochromanol metabolism not inhibitable by sesamin existed in A549 cells, postincubation recovery of substrate (-TOH or -T3) was determined in the presence and absence of sesamin. Sesamin treatment resulted in an approximate doubling in increase in cell-associated -TOH and an approximate 4 times increase in cell-associate -T3 (Fig. 6B). These increases in cell-associate substrate were accompanied by increases in unmetabolized substrate recovered in the media at the end of the incubation period. The total mass of added -T3 recovered after 48 h in the absence and presence of sesamin was 54 and 92%, respectively, of that in culture dishes without cells.

    Evaluation of TOH--oxidation activity in rat primary type II pneumocytes and H69AR cells. The finding that type II pneumocyte-like A549 cells exhibited TOH--hydroxylase activity prompted us to undertake a similar evaluation of primary type II pneumocytes isolated from rat lung using 25 µmol/L - or -TOH as substrates. These cultures did not exhibit detectable enzyme activity. We also evaluated a second human epithelial lung cancer cell line, H69AR, originally isolated from a human small-cell carcinoma, to determine if expression of TOH--hydroxylase activity was a general characteristic of lung cancer cells. These cells also did not exhibit detectable activity.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We previously demonstrated that human hepatoblastoma (HepG2) cultures, human liver microsomes, and recombinant human CYP4F2 catalyze -hydroxylation of TOHs at one of the terminal phytyl methyl groups, with -TOH a much better substrate than -TOH (1,7). The initial -hydroxylation is followed by dehydrogenation to the terminal (13') carboxychromanol and truncation of the phytyl tail by alternating removal of two- or three-carbon moieties, ultimately yielding the water-soluble 3'-carboxychromanol metabolites previously shown to be excreted in urine (9–12). The TOH--oxidation pathway may provide certain cell types a means of regulating their vitamin E status. Yamane and Abe (3) reported that the human cell lines A549 (lung adenocarcinoma) and HepG2 (hepatoblastoma) expressed CYP4F2 mRNA, and that A549 cells exhibited greater leukotriene B4--hydroxylase activity (attributed to CYP4F2) than HepG2 cells. Therefore we sought to ascertain whether A549 cultures exhibited TOH--hydroxylase activity, and if so, whether the activity exceeded that of HepG2 cells, as expected if CYP4F2 was responsible for TOH--hydroxylase activity in these 2 cell types.

Consistent with this hypothesis, A549 cultures metabolized both TOHs and T3s with TOH--hydroxylase activity and produced the array of side-chain -oxidation products previously observed in HepG2 cultures. In addition, A549 cells metabolized all TOH and T3 substrates more extensively than HepG2 cells (Fig. 3), a finding that could not be explained on the basis of differential substrate uptake by the 2 cell types. However, the difference in -TOH metabolism between 2 cell types was not statistically significant. In a comparison of TOH--hydroxylase activity of cell-free homogenates, A549 homogenates exhibited 2–5 times more activity than HepG2 cells. This is consistent with the finding of Yamane and Abe (3) who reported 2 times more leukotriene B4 hydroxlyase specific activity in A549 homogenates than in HepG2 cells, an activity attributed to CYP4F2. Thus the difference in activity of intact cells is likely due to elevated enzyme protein in A549 cells relative to HepG2 cells. In addition, the difference in activity appears to be due to greater CYP4F2 activity in the nuclear envelope of A549 cells, because removal of the nuclear fraction of the homogenate resulted in lower and equivalent activity between the 2 cell types.

A novel and unexpected finding was that the major metabolite of - and -TOHs and T3s in A549 cultures was the 9'-carboxychromanol. This ionized but relatively hydrophobic intermediate did not accumulate within the cells but was efficiently secreted into the media. The 9'-carboxychromanols accumulated in only small amounts in HepG2 cultures, apparently because of truncation of this intermediate to 5'- and 3'-carboxychromanols. We investigated 2 potential causes of the marked accumulation of 9'-carboxychromanols by A549 cultures. The first was the potential inability of A549 cells to take up and further metabolize 9'-carboxychromanols once secreted into the culture medium. This possibility was not supported by the finding that A549 cells (and HepG2 cultures) incubated with 9'--(3'-en)carboxychromanol-enriched media, produced by a separate A549 culture, synthesized 5'--carboxychromanol at the expense of this substrate. The second possibility was that accumulation of 9'-carboxychromanols by A549 cultures reflected a limited capacity of its conversion to 7'-carboxychromanols in this cell type relative to that of HepG2 cells. This possibility was supported by the findings that HepG2 cultures incubated with an elevated substrate concentration that yielded an overall rate of metabolism similar to that of A549 cultures still did not accumulate 9'-carboxychromanols, whereas reducing flux through this pathway in A549 cultures by treatment with sesamin reduced the accumulation of 9'-carboxychromanols and resulted in a metabolite pattern similar to that of HepG2 cells. Therefore A549 cells may prove valuable in probing the details of the -oxidation process that occur downstream of the initial CYP-mediated -hydroxylation event.

We observed marked differences in rates of metabolism between TOHs and T3s by A549 cells (Fig. 3). However, certain cell types are known to take up different tocochromanols to different extents (13). To investigate whether the vitamer differences was due solely to differential substrate uptake, we compared the time course of elimination of various tocochromanols in A549 cultures previously loaded with similar intracellular concentrations of the substrates. Under these conditions, -T3 was cleared rapidly and -TOH was cleared slowly. Only minor and similar amounts of unchanged substrates were recovered in the medium, illustrating those differential rates of catabolism, rather than substrate uptake or secretion, was largely responsible for the observed differences in tocochromanol metabolism in cultures continually exposed to substrate.

Sesamin and ketoconazole exhibited remarkably similar and potent inhibition of tocochromanol metabolism by A549 cells. Nearly complete inhibition was observed even at the modest concentration of 5 µmol/L. The similarity of inhibition is supportive of a single CYP responsible for -oxidation of tocochromanols in these cells, because it is unlikely that different enzymes would exhibit identical extents of inhibition by both substances. Furthermore, substrate recovery in the presence and absence of sesamin indicated no evidence of other pathways of tocochromanol metabolism by these cells, because inhibition of metabolism resulted in the accumulation of unchanged substrates in cells and medium. Sesamin has also been reported to inhibit certain fatty acid desaturases but with IC₅₀ (Inhibitory Concentration, concentration resulting in 50% inhibition) values above 250 µmol/L in cell culture (14,15), i.e., 2 orders of magnitude above that of inhibition of tocochromanol metabolism.

The A549 cell line was derived from a human lung adenocarcinoma of lung type II cell origin (4), and A549 cells exhibit many structural and biochemical features characteristics of type II alveolar pneumocytes. Therefore we sought to determine if the expression of TOH--hydroxylase activity in A549 cells reflected a characteristic of type II pneumocytes, or rather, arose as a consequence of the transformation process. Primary type II pneumocytes isolated from rat lung did not exhibit detectable TOH--hydroxylase activity, nor did H69AR cells, a cell line of human small-cell carcinoma origin. These findings suggest that TOH--hydroxylase activity of A549 cells is probably a consequence of the transformation of normal type II cells to cancer cells but not of transformation of all lung cell types.

Jiang et al. (16) reported that eicosanoid synthesis by A549 cells could be inhibited by -TOH and its 3'-carboxychromanol metabolite but not by -TOH, when these agents were added to A549 cultures. These studies were conducted without the knowledge that A549 cells metabolize -TOH and that the predominant metabolite was 9'--carboxychromanol. Thus the reported inhibition of cyclooxygense activity by -TOH in the cells may have been due to this long-chain metabolite rather than to -TOH per se. A549 epithelial cells may be useful for the investigation of the biological activity of long-chain carboxychromanols and for biosynthesis of 9'-carboxychromanols for evaluation of activities in other experimental systems.

	ACKNOWLEDGMENTS

 
The authors thank Sandra Bates, Institute of Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, for isolation and incubation of primary type II pneumocytes.

	FOOTNOTES

 
¹ Presented in part at EB2004, April 17–21, 2004, Washington, DC [You, C.-S., McCormick, C. C. & Parker, R. S. (2004) Metabolism and cytotoxicity of TOHs and T3s in human type II (A549) pneumocytes. FASEB J. 18(4): A159 (abs.)].

² This work was supported by USDA/NRI 9800692, and by National Institutes of Health Training Grant DK07158–25 (T.J.S).

⁴ Abbreviations used: CYP, cytochrome P450; d₆--TOH, deuterated -TOH; FBS, fetal bovine serum; T3, tocotrienol; TOH, tocopherol.

Manuscript received 20 July 2004. Initial review completed 12 August 2004. Revision accepted 21 October 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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