␥-Tocopherol and its major metabolite, in contrast to ␣-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells Qing Jiang, Ilan Elson-Schwab, Chantal Courtemanche, and Bruce N. Ames* Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; and Children’s Hospital Oakland Research Institute, 5700 M. L. King Jr. Way, Oakland, CA 94609 Contributed by Bruce N. Ames, July 28, 2000 Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin substituted 5-position, ␥T is a better nucleophile and detoxifies E2 (PGE2) plays a key role in inflammation and its associated NOx by forming a stable adduct, 5-nitro-␥T (17–19). In an in vitro diseases, such as cancer and vascular heart disease. Here we report system, ␥T, compared with ␣T, exhibits stronger inhibition of ␥ ␥ that -tocopherol ( T) reduced PGE2 synthesis in both lipopolysac- lipid peroxidation induced by peroxynitrite (17). Dietary ␥Tis charide (LPS)-stimulated RAW264.7 macrophages and IL-1␤- primarily metabolized to 2,7,8-trimethyl-2-(␤-carboxyethyl)-6- treated A549 human epithelial cells with an apparent IC50 of 7.5 hydroxychroman (␥-CEHC), a water-soluble compound found and 4 ␮M, respectively. The major metabolite of dietary ␥T, in human urine and possessing natriuretic activity (20, 21). 2,7,8-trimethyl-2-(␤-carboxyethyl)-6-hydroxychroman (␥-CEHC), We have recently observed that ␥T supplementation leads to Ϸ ␮ also exhibited an inhibitory effect, with an IC50 of 30 M in these the inhibition of protein nitration and ascorbate oxidation, and cells. In contrast, ␣-tocopherol at 50 ␮M slightly reduced (25%) spares vitamin C in rats with zymosan-induced peritonitis (Q.J., PGE2 formation in macrophages, but had no effect in epithelial J. Lykkesfeldt, E. T. Shigeno, B.N.A., M. K. Shigenaga, and S. cells. The inhibitory effects of ␥T and ␥-CEHC stemmed from their Christen, unpublished data). In addition to its reactivity toward inhibition of COX-2 activity, rather than affecting protein expres- NOx, it appears that ␥T plays a role in defending against sion or substrate availability, and appeared to be independent of inflammation-related damage. In the present study, we investi- ␥ antioxidant activity. -CEHC also inhibited PGE2 synthesis when gated the effects of ␥T on the inflammatory response in mac- exposed for1htoCOX-2-preinduced cells followed by the addition rophages and human epithelial cells. We found that ␥T inhibited of arachidonic acid (AA), whereas under similar conditions, ␥T the generation of prostaglandin E2 (PGE2), an important me- required an 8- to 24-h incubation period to cause the inhibition. The diator synthesized via the cyclooxygenase-2 (COX-2)-catalyzed ␥ ␥ inhibitory potency of T and -CEHC was diminished by an increase oxidation of arachidonic acid (AA) during inflammation. Our in AA concentration, suggesting that they might compete with AA results show that both ␥T and its major hydrophilic metabolite, at the active site of COX-2. We also observed a moderate reduction ␥-CEHC, at physiological concentrations are effective in inhib- of nitrite accumulation and suppression of inducible nitric oxide iting COX-2 activity in intact cells, whereas ␣T is much less ␥ synthase expression by T in lipopolysaccharide-treated macro- effective. phages. These findings indicate that ␥T and its major metabolite possess anti-inflammatory activity and that ␥T at physiological Materials and Methods concentrations may be important in human disease prevention. Materials. ␣T (99%) and ␥T (95–97%) were purchased from Acros Organics (Summerville, NJ) or Fluka. ␥-CEHC (Ն98%) nflammatory diseases affect millions of people in the world, and lipid hydroperoxide-free AA were from Cayman Chemicals Iand chronic inflammation is one of the major contributors to (Ann Arbor, MI). 2Ј,7Ј-Dichlorofluorescin (DCFH) was from the development of cancer as well as neurodegenerative and Molecular Probes. Tissue culture reagents were all from cardiovascular diseases (1, 2). Antioxidant vitamins, which de- GIBCO͞BRL. Bacterial lipopolysaccharide (LPS; B E. coli fend against oxidants such as those produced during inflamma- 055:B5) was from Difco. IL-1␤ and all other chemicals were from tion, are believed to play an important role in public health and Sigma. human disease prevention (2). Among these vitamins, ␣-tocoph- erol (␣T), the predominant form of vitamin E in many tissues Cell Culture. Murine RAW264.7 macrophages were routinely and the exclusive component in most vitamin E supplements, has cultured in DMEM containing 10% FBS. Human epithelial been extensively studied both in vitro and in vivo (3, 4). In cancer cells (A549) were obtained from American Type Culture contrast, ␥-tocopherol (␥T), while constituting 70–80% of vita- Collection and cultured in F12K medium supplemented with min E in U.S. diets (5), was mostly ignored in the past because 10% FBS. of its relatively low animal plasma and tissue concentrations (6, 7), which result in a poor bioactivity as defined by the rat fetal Cellular Uptake of ␣T and ␥T. Cells were incubated in DMEM resorption assay (8). containing 0.5% FBS supplemented with 10 ␮M ␣Tor␥T for However, emerging evidence indicates that ␥T may be impor- 14 h. After harvested by scraping, cells were washed twice with tant in the defense against degenerative diseases. Some epide- miological studies suggest that high intakes or high plasma levels of ␣T predict low incidence of heart disease (9, 10), whereas Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; DCFH, 2Ј,7Ј-dichlorofluorescin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NOx, reactive nitrogen oxide others fail to observe the inverse correlation (11, 12) or pro- ␣ ␣ ␥ ␥ ␣ species; PGE2, prostaglandin E2; PGD2, prostaglandin D2; T, -tocopherol; T, -tocoph- tective effects from T supplementation (13, 14). Instead, erol; ␥-CEHC, 2,7,8-trimethyl-2-(␤-carboxyethyl)-6-hydroxychroman. several independent investigations (11, 12, 15) demonstrate that *To whom reprint requests should be addressed. E-mail: [email protected]. plasma concentrations of ␥T, but not ␣T, are inversely correlated The publication costs of this article were defrayed in part by page charge payment. This to the incidence of coronary heart diseases. Cooney et al. (16) article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. and our laboratory (17) have shown that ␥T is superior to ␣Tin §1734 solely to indicate this fact. trapping reactive nitrogen oxide species (NOx), mutagenic elec- Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.200357097. trophiles generated during inflammation. Because of the non- Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.200357097 11494–11499 ͉ PNAS ͉ October 10, 2000 ͉ vol. 97 ͉ no. 21 Downloaded by guest on September 29, 2021 Hanks’ balanced salt solution. ␣T and ␥T were measured by brane were detected by chemiluminescent kit (Roche Molecular HPLC by using electrochemical detection. The amounts of ␣T Biochemicals) and quantified by IMAGEQUANT. and ␥T in macrophages were 0.87 Ϯ 0.12 and 1.05 Ϯ 0.1 nmol͞106 cells, and in epithelial cells were 0.90 Ϯ 0.1 and 1.25 Ϯ Statistical Analysis. The unpaired Student’s t test was used in the 0.2 nmol͞106 cells, respectively. statistical analysis. All results are expressed as means Ϯ SDs. PGE2 Generation During Chronic LPS and IL-1␤ Treatment. RAW264.7 Results ϫ 5 cells (5 10 per well) were allowed to attach overnight in a Differential Effects of ␣T, ␥T, and ␥-CEHC on PGE2 Generation in 24-well plate. Confluent cells were incubated with DMEM RAW264.7 Cells Stimulated with LPS and Epithelial Cells Treated with ␤ containing 0.5% FBS with ethanol (control) or tocopherols for IL-1 . A marked increase of PGE2 generation was observed in the indicated time and then 0.1 ␮g͞ml LPS was introduced for RAW264.7 macrophage when cells were treated with 0.1 ␮g͞ml 14 h. Similarly, confluent A549 cells were incubated with LPS. Supplementation with ␥T led to a dose-dependent reduc- Ϯ DMEM containing 0.5% FBS supplemented with ethanol or tion of PGE2 synthesis (Fig. 1A), with an apparent IC50 of 7.5 various tocopherols for 8 h and then treated with 10 ng͞ml IL-1␤ 2 ␮M. In contrast to ␥T, ␣T with concentrations of less than 10 ␮ ␮ for 24 h, at which time the medium was collected and PGE2 M had little effect on PGE2 production, whereas 10 M generation was measured. ␥-CEHC showed Ϸ 20% inhibition. At 50 ␮M, ␥T and ␥-CEHC led to 70–80% and 60% reduction of PGE2 synthesis, respec- COX-2 Activity in Intact Cells. A549 cells were pretreated with 10 tively, whereas ␣T showed only Ϸ25% inhibition. ͞ ␤ ng ml IL-1 for 24 h, then incubated with fresh medium A549, a human lung epithelial cancer cell line, has been used containing tocopherols or ethanol for the indicated time, and to evaluate the anti-inflammatory effect of nonsteroidal anti- finally incubated with exogenous AA for 10 min. PGE2 release inflammatory drugs in intact cells (24). In IL-1␤-treated A549 was measured as an index of COX-2 activity. cells, both ␥T and ␥-CEHC exhibited dose-dependent inhibition Ϯ ␮ Ϯ ␮ of PGE2 release, with IC50 of 4 1 M and 30 3 M, Nitrite Measurement and DCFH Oxidation. Formation of NOx was respectively (Fig. 1B). On the other hand, ␣T did not show any determined by nitrite accumulation in LPS-activated macro- effect even at a concentration of 40 ␮M. The inhibitory effect phages by using the Griess assay (22). The generation of total of ␥T was also observed in the presence of exogenous AA reactive oxygen species was evaluated by the oxidation of (Fig. 1C). nonfluorescent DCFH to the highly fluorescent 2Ј,7Ј- Maximum inhibition of PGE2 was achieved when cells were dichlorofluorescein, as monitored by the increase in fluores- preincubated with ␥Tor␣T for 8–14 h before LPS or IL-1␤ cence intensity at 520 nm with excitation at 485 nm (23), by using treatment, which is likely attributable to their slow cellular a Cytofluor 2350 fluorescent measurement system (Millipore).