Biochemical and Biophysical Research Communications 277, 531534 (2000) doi:10.1006/bbrc.2000.3706, available online at http://www.idealibrary.

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Cytochrome P4503A-Dependent Metabolism of Tocopherols and Inhibition by Sesamin

Robert S. Parker, 1 Timothy J. Sontag, and Joy E. Swanson
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853

Received September 21, 2000

Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specic cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 M), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of - and -tocopherol in rat primary hepatocytes, and metabolism of - and -tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 M. These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the rst evidence of a specic enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism. 2000 Academic Press Key Words: tocopherols; metabolism; cytochrome P450; carboxychroman; sesame; sesamin; ketoconozole.

Tissue concentrations of tocopherols and tocotrienols, which collectively constitute the vitamin E family of compounds, is inuenced by both dietary intake and their rates of elimination. The pathways of elimination of vitamin E, and the nature of the biotransformation reactions which may facilitate their elimination, are poorly understood. The major dietary tocopherols are metabolized at least in part to water-soluble carboxychromans which are excreted in the urine (1 4). These urinary metabolites apparently arise via oxidation of the phytyl side chain followed by its truncation to short
Support provided by USDA/NRI and NIH Training Grant DK07158-25 (T.J.S.). 1 To whom correspondence should be addressed. Fax: (607) 2551033. E-mail: rsp3@cornell.edu.

chain carboxychromans, while the chromanol head group remains intact. A substantial proportion of estimated daily intake of -tocopherol can be accounted for by urinary excretion of 3--carboxychroman (5). We have recently reported that human hepatoblastoma cells metabolize -tocopherol to 3- and 5-carboxychromans and that both are excreted in human urine (6). While an initial P450-mediated oxidation is likely to be involved in this metabolic pathway, none of the enzyme(s) responsible for side chain truncation have been elucidated. The extent to which catabolism of tocopherols to carboxychromans is an important determinant of tissue concentrations of tocopherols, and the extent to which this may be inuenced by other dietary components, is not known. It has been reported that rats fed sesame seed, or the puried sesame lignans sesamin or sesaminol, exhibit elevated plasma and tissue levels of tocopherols, particularly -tocopherol (79). This observation can potentially be explained by inhibition of tocopherol catabolism by these furofuran lignans. The observation that methylenedioxyphenyl lignans can act as insecticide synergists (10, 11) supports this hypothesis. Synergists, including some methylenedioxyphenyl compounds, commonly exert their effect through inhibition of the P450(s) responsible for the detoxication of insecticides, thus increasing the halife, tissue concentration, and toxicity of the latter (10, 12). Here we report evidence for the involvement of cytochrome P4503A, the major P450 isoform family in human liver, in the oxidative catabolism of tocopherols to carboxychromans. We further demonstrate that the sesame lignan sesamin is a potent inhibitor of this pathway, suggesting a mechanism of the tocopherolenhancing effect of dietary sesame seed and its furofuran lignans. MATERIALS AND METHODS
Cell culture. HepG2 cells (C3A subclone CRL-10741, American Type Culture Collection, Manassas, VA) were maintained in Minimal Essential Media (MEM; Atlanta Biologicals, Atlanta, GA) sup0006-291X/00 $35.00
Copyright 2000 by Academic Press All rights of reproduction in any form reserved.

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plemented with Earles salts, sodium bicarbonate, and 10 percent fetal bovine serum (FBS), without antibiotics. Cells were grown in 60 mm polypropylene dishes at 37C with 3 ml media under 5% CO 2/ 95% air, and used only after they had reached conuence. FBS enriched with -, -, or -tocopherol was prepared by dropwise addition of 0.25 or 0.5 moles of RRR--tocopherol or RRR--tocopherol (99%, Fluka Biochemicals, Milwaukee, WI) or RRR--tocopherol (Henkel, Inc., La Grange, IL), dissolved in a small volume of absolute ethanol (30 or 60 l per ml FBC), while vortexing. The tocopherolenriched FBS was allowed to stand overnight at 4C before diluting 1:10 with MEM. The resulting complete medium contained 25 or 50 mol/L tocopherol. Stock solutions of the test P450 inhibitors ketoconozole, and sulphaphenzole (Sigma Chemical Co., St. Louis), and of puried sesamin (a gift from N. G. Lewis, Washington State University, Pullman, WA), were prepared in absolute ethanol. Just prior to use an appropriate volume of these solutions were added to FBS, diluted 1:10 with MEM, and applied to the culture dishes for a 4 h preincubation period. Cyclosporin (Sigma) was prepared in demethylsulphoxide and added to complete media for a similar preincubation period. The media was then replaced with 3 ml tocopherolcontaining media, the test compounds replaced, and the cells incubated at 37C. After 24 or 48 h the medium was collected, and the cells were washed twice with cold phosphate-buffered saline and scraped from the plate. Both media samples and cell pellet suspensions were frozen at 40C under argon until extracted. Primary rat hepatocytes were prepared from male SpragueDawley rats by collagenase perfusion (13). The perfusate contained 1 mM aminoguanidine to promote maintenance of cytochrome P450 levels (14). The cells were washed twice with phosphate-buffered saline and collected by low speed centrifugation. Cells were resuspended in Williams E medium and approximately 800,000 cells were introduced into 100 mm collagen-coated culture dishes and allowed to attach for 4 h. Ketoconozole (or the same volume of ethanol as vehicle control) was then added. Four hours later the medium was replaced with - or -TOH-enriched medium, the ketoconozole replaced, and the cells incubated for an additional 24 or 48 h. The medium was then collected and frozen for later analysis of - and -carboxychroman metabolites. Quantication of carboxychroman metabolites and cell-associated tocopherol. Concentrations of 3- and 5-carboxychroman metabolites (carboxyethylhydroxychroman, or CEHC, and carboxymethylbutylhydroxychroman, or CMBHC, respectively) of -, -, and -tocopherol in cell culture media were determined by gas chromatography-mass spectrometry (GC/MS) using a deuterium-labeled analogs of - and -CEHC essentially as described earlier (5, 6). The GC/MS system consisted of a Hewlett Packard 5890 GC coupled to a Hewlett Packard 5972 mass selective detector (MSD). The GC was equipped with a Hewlett Packard HP-1 methylsiloxane capillary column (30 m 0.25 mm), operated in split injection mode (20:1) using helium as the carrier gas (1 ml/min) and an injection volume of 1 l. The oven temperature was programmed to ramp from 200C (2 min hold) to 210C at 20C/min (5 min hold), to 230C at 25C/min (6 min hold), then to 280C at 25C/min (nal hold 8 min). In selected ion mode (SIM) the following ions were monitored: m / z 408 (unlabeled -CEHC), 410 (d 2--CEHC, internal standard), 450 (CMBHC). In scan mode, the ion range monitored was m / z 100 to 550 at 1.8 scans/s. Disilyl-d 2--CEHC peak areas were corrected for natural abundance contributions by heavy isotopes of carbon and silicon present in the unlabeled -CEHC metabolite. The media concentration of -CEHC and -CMBHC were calculated using the corrected d 0 / d 2 peak area ratios and the concentration of the d 2 --CEHC internal standard. Concentrations of -CEHC and -CMBHC were determined similarly using a custom-synthesized d 9 --CEHC internal standard prepared by deuteriomethylation of the carboxybenzopyran obtained by reaction of hydroquinone with -methyl--vinylbutyrolactone. Details of this synthesis will be reported separately. The concentrations of 3- and 5--carboxychroman metabolites were determined using the d 2 --CEHC internal standard without correct-

FIG. 1. Inhibition of tocopherol metabolism by ketoconozole in HepG2/C3A and rat primary hepatocyte cultures. HepG2/C3A cells or primary rat hepatocytes were incubated with media supplemented with 25 M - or -tocopherol in the absence (open columns) or presence (shaded columns) of 1.0 M ketoconozole, as described under Materials and Methods. Concentrations of the corresponding 3- and 5-carboxychroman metabolites (CEHC and CMBHC, respectively) in the culture media was determined after 48 h of incubation. Metabolite concentrations were expressed relative to cell protein (HepG2/C3A cells) or cell DNA (rat hepatocytes).

ing for potential differences in detector response, as synthetic standards for the -TOH metabolites are not yet available. Preliminary experiments indicated that the cells do not accumulate these metabolites, and consequently metabolite analyses were carried out in media samples only. Aliquots of washed cells were extracted with hexane after addition of d 9 --tocopherol as internal standard and protein precipitation with one volume of cold absolute ethanol. Concentrations of cellassociated tocopherols were determined by GC/MS using this internal standard (15) and expressed relative to cell protein, accounting for the relative detector responses of - and -tocopherol.

RESULTS The inuence of ketoconozole on metabolism of -tocopherol to its corresponding 3- and 5-carboxychroman metabolites by HepG2/C3A cells is shown in Fig. 1. Ketoconozole, a potent and specic inhibitor of the CYP3A family of P-450 isozymes (16, 17), inhibited -tocopherol metabolism by over 90 percent when present in the culture medium at 1.0 M. Lower concentrations were also effective in C3A cultures, yielding approximately 50 percent inhibition at 0.25 M. Sulphaphenazole, a known inhibitor of CYP2C family of P-450 isozymes (16, 17) was without effect at 1 and 10 M (data not shown). Likewise, there was no effect of cyclosporin, an inhibitor of CYP27, an isoform known to catalyze the oxidation of the isoprenoid side chain of cholesterol (18). Ketoconozole exhibited no effect on cell growth as reected by cell protein accumulation. To demonstrate that the potent inhibitory effect of ketoconozole on tocopherol metabolism is not restricted to tumor cells, we repeated the experiment using freshly cultured rat primary hepatocytes. Ketoconozole (1 M) was effective in inhibiting metabolism

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FIG. 2. Inhibition of -tocopherol metabolism by ketoconozole in HepG2/C3A cultures. HepG2/C3A cells were incubated with media supplemented with 25 M -tocopherol in the absence (open columns) or presence (shaded columns) of 1.0 M ketoconozole, as described under Materials and Methods. Concentrations of 3- and 5--carboxychroman metabolites (CEHC and CMBHC, respectively) in the culture media were determined after 48 h of incubation and expressed relative to cell protein.

of both - and -tocopherol by these primary hepatocyte cultures, as illustrated in Fig. 1. Metabolism of both tocopherols to their corresponding carboxychromans was reduced by approximately 90 percent over the 48-h culture period in the presence of 1 M ketoconozole. Similarly low concentrations of ketoconozole (1.0 and 0.25 M) were also effective in blocking metabolism of -tocopherol to its 3- and 5--carboxychroman metabolites by HepG2/C3A cultures, as illustrated in Fig. 2. The effect of sesamin, a major sesame seed lignan, on -tocopherol metabolism by HepG2/C3A cells is illustrated in Fig. 3. Sesamin (1 M) reduced -carboxychroman synthesis by approximately 90 percent over the 48 h incubation period without any effect on cell growth. Tocopherol analysis of washed cells indicated slightly higher concentrations of cell-associated tocopherol in the presence of ketoconozole or sesamin (data not shown). DISCUSSION CYP3A is the major family of cytochrome P-450 isoforms present in mammalian liver, with the CYP3A4 isoform constituting approximately 30 percent of total hepatic cytochrome P-450 in human liver (17). In fetal liver and liver tumor cells, CYP3A7 is a major P-450 isoform, while the adult CYP3A4 isoform is absent (19). The HepG2/C3A cell line is a subclone of the HepG2 line, a well differentiated human hepatoblastoma line derived from a hepatocellular carcinoma (20). HepG2 cells have been well characterized with respect to their cytochrome P-450 expression and activity. These cells express CYP3A7 but not CYP3A4 (19), and we previously reported that these cells actively metabolize -tocopherol but not -tocopherol (6).

In rat liver the mature isoforms of the 3A family are CYP3A1 and CYP3A2. Ketoconozole, an imidizole antifungal agent, is a well characterized and highly specic non-competitive inhibitor of CYP3A isoforms when used at low micromolar concentrations. This and other P-450 inhibitors are commonly used to indicate the involvement of specic P-450 enzymes in substrate metabolism (16, 17). Here we show that ketoconozole effectively blocks the phytyl side-chain shortening of tocopherols to the corresponding water-soluble carboxychroman metabolites also excreted in human urine, demonstrating the essential role of CYP3A in this process. Sulphaphenazole, a potent inhibitor of the CYP2C family of P-450 enzymes, and cyclosporin, an inhibitor of CYP27-catalyzed terminal hydroxylation of the isoprenoid side chain of cholesterol in bile acid synthesis, were without effect. CYP3A-dependent sidechain metabolism of tocopherols was demonstrated in both human hepatoblastoma cells and in rat primary hepatocytes, the latter for which it is reported for the rst time actively metabolize both - and -tocopherol to their corresponding 3 and 5 carboxychromans. The very limited ability of HepG2/C3A cells to metabolize -tocopherol (relative to - or -tocopherol) suggests that the mature isoforms of CYP3A can oxidize all tocopherols while the fetal form exhibits substrate preference for the non- tocopherols. These data constitute the rst evidence of a specic enzyme relevant to vitamin E metabolism in any species, and implicates the major human liver cytochrome P-450 isoform in the catabolism of tocopherols to the water-soluble metabolites previously reported in human urine. Sesamin is a furofuran lignan and major nonsaponiable lipid in sesame seed and sesame seed oil (21). When fed to rats, sesame seeds, sesame seed oil, or puried sesamin result in elevated concentrations of

FIG. 3. Inhibition of -tocopherol metabolism by sesamin in HepG2/C3A cultures. HepG2/C3A cells were incubated with media supplemented with 25 M -tocopherol in the absence (open columns) or presence (shaded columns) of 1.0 M sesamin, as described under Materials and Methods. Concentrations of the corresponding 3- and 5--carboxychroman metabolites (CEHC and CMBHC, respectively) in the culture media were determined after 48 h of incubation and expressed relative to cell protein.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 7. Yamashita, K., Nohara, Y., Katayama, K., and Namiki, M. (1992) Sesame seed lignans and -tocopherol act synergistically to produce vitamin E activity in rats. J. Nutr. 122, 2440 2446. 8. Yamashita, K., Lizuka, Y., Imai, T., and Namiki, M. (1995) Sesame seed and its lignans produce marked enhancement of vitamin E activity in rats fed a low -tocopherol diet. Lipids 30, 1019 1028. 9. Kamal-Eldin, A., Pettersson, D., and Appelqvist, L-A. (1995) Sesamin (a compound from sesame oil) increases tocopherol levels in rats fed ad libitum. Lipids 30, 499 505. 10. Casida, J. E. (1970) Mixed-function oxidase involvement in the biochemistry of insecticide synergists. J. Agr. Food Chem. 18, 753772. 11. MacRae, W. D., and Towers, G. H. N. (1984) Biological activities of lignans. Phytochem. 23, 12071220. 12. Doud, P. F., Smith, M. C., and Sparks, T. C. (1983) Detoxication of plant toxins by insects. Insect Biochem. 13, 453 462. 13. Berry, M., Edwards, A., and Borritt, G. (1991) Isolated hepatocytes: Preparation, properties, and applications, pp. 16 57, Elsevier, New York, NY. 14. Lopez-Garcia, M. (1998) Endogenous nitric oxide is responsible for the early loss of P450 in cultured rat hepatocytes. FEBS Lett. 438, 145149. 15. Burton, G. W., and Daroszewska, M. (1996) Deuterated vitamin E: Measurement in tissues and body uids. In Free Radicals: A Practical Approach (Punchard, N. A., and Kelly, F. J., Eds.), Oxford University Press, Oxford, UK. 16. Rendic, S., and Di Carlo, F. J. (1997) Human cytochrome P450 enzymes: A status report summarizing their reactions, substrates, inducers, and inhibitors. Drug. Met. Rev. 29, 413580. 17. Pelkonen, O., Maenpaa, J., Taavitsainen, Pl., Rautio, A., and Raunio, H. (1998) Inhibition and induction of human cytochrome P450 (CYP) enzymes. Xenobiotica 28, 12031253. 18. Winegar, D., Salisbury, J., Sundseth, S., and Hawke, R. (1996) Effects of cyclosporin on cholesterol 27-hydroxylation and LDL receptor activity in HepG2 cells. J. Lipid Res. 37, 179 191. 19. Schuetz, E. G., Schuetz, J. D., Strom, S. C., Thompson, M. T., Fisher, R. A., Molowa, D. T., Li, D., and Guzelian, P. S. (1993) Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes. Hepatology 18, 1254 1262. 20. Javitt, N. (1990) HepG2 cells as a resource for metabolic studies: Lipoprotein, cholesterol, and bile acids. FASEB J. 4, 161168. 21. Kamal-Eldin, A., and Appelqvist, L. A. (1994) Variations in the composition of sterols, tocopherols, and lignans in seed oils from four Sesamum species. J. Am. Oil Chem. Soc. 71, 149 156.

tocopherols in plasma and tissues (79). This effect has been attributed to a putative antioxidant sparing effect of sesamin or a sesamin metabolite. Here we demonstrate that sesamin is a potent inhibitor of the catabolism of -tocopherol to -carboxychromans. Inhibition of tocopherol catabolism by sesamin (or ketoconozole) was accompanied by a slight increase in cell-associated tocopherol, demonstrating that the inhibitory effect was not due to impairment of tocopherol uptake by these cells. Thus the likely mechanism explaining the effect of sesamin on tissue tocopherol levels is inhibition of CYP3A-dependent tocopherol catabolism, resulting in enhanced accumulation of tocopherols in plasma and tissues. ACKNOWLEDGMENTS
The authors thank Mr. Larry Hirschberger for valuable assistance in the preparation of primary rat hepatocytes, and Dr. Norman Lewis, Institute of Biological Chemistry, Washington State University, for providing the sesamin used in these studies.

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