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Diethanolamine Inhibits Choline Uptake and Phosphatidylcholine Synthesis in Chinese Hamster Ovary Cells
Biochemical and Biophysical Research Communications 262, 600 – 604 (1999) Article ID bbrc.1999.1253, available online at http://www.idealibrary.com on
Diethanolamine Inhibits Choline Uptake and Phosphatidylcholine Synthesis in Chinese Hamster Ovary Cells Lois D. Lehman-McKeeman 1 and Elizabeth A. Gamsky Human and Environmental Safety Division, Procter and Gamble Co., P.O. Box 538707, Cincinnati, Ohio 45253
Received July 26, 1999
Diethanolamine (DEA), an alkanolamine used widely in industry, is hepatocarcinogenic in mice. The goal of this work was to determine whether DEA altered choline homeostasis in cultured cells, so as to ascertain whether the liver tumor response may be related to choline deficiency. CHO cells were cultured in Ham’s F-12 medium containing DEA (0-1000 mg/ml) and [ 33P]-phosphorus was used to label phospholipid pools. After 48 hours incubation, lipids were extracted and [ 33P]-labeled phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) accounted for 51 6 0.7% of the total lipid 33P incorporation. DEA had no effect on cell number or total phospholipid biosynthesis, but it significantly decreased the incorporation of 33P into PC at concentrations >50 mg/ml. DEA (>20 mg/ml) also inhibited the uptake of [ 3H]-choline into CHO cells, with 95% inhibition observed at 250 mg/ml. To determine whether supplemental choline prevented PC synthesis inhibition by DEA, CHO cells were cultured with or without excess choline (30 mM) and DEA (500 mg/ml). DEA reduced PC synthesis to 27 6 3% of total phospholipids, but had no effect on PC synthesis in choline-supplemented cells. When [ 14C]-DEA was incubated with CHO cells, it was also incorporated into the phospholipid fraction. Collectively, these results indicate that DEA reversibly inhibits PC synthesis by blocking choline uptake and competing for utilization in the CDP-choline pathway in CHO cells. © 1999 Academic Press
Choline is an endogenous compound and dietary component that is essential for the normal function of all cells (1– 4). This quaternary amine is present in tissues primarily as phosphatidylcholine (PC), which accounts for about one half of the total phospholipid 1 Corresponding author. Fax: (513) 627-1908. E-mail: lehman [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
content of mammalian cells (5, 6). Other important choline-containing metabolites include acetylcholine, platelet-activating factor, choline plasmalogens, phosphocholine and betaine (4). In most cells, choline uptake is the first step in the cytidyl diphosphate (CDP)-choline pathway for the biosynthesis of choline-containing phospholipids (7, 8). Once inside the cell, choline is phosphorylated by choline kinase, after which phosphocholine is converted to CDP-choline by CTP-cholinephosphate cytidyltransferase, the regulated, rate-limiting step in PC synthesis (9, 10). CDP-choline in combination with diacylglycerol forms PC and cytidine monophosphate. It is generally recognized that choline is essential for cells because intentional deprivation of choline from cell culture medium disrupts cell growth and division (5, 11–13), and dietary choline deficiency alters hepatic (4, 14) and renal (4, 15) function. Moreover, choline deficiency is the only single-nutrient deficiency that causes spontaneous carcinogenesis in rodents (4, 16 –18). Diethanolamine (DEA), an alkanolamine structurally similar to ethanolamine, is widely used in industry and its fatty acid condensates are present in many household products. Recently, it has been shown that with lifetime exposure, DEA causes liver tumors in mice (19). However, DEA showed no evidence of DNA reactivity, suggesting that a secondary, non-genotoxic mechanism may be involved in the hepatocarcinogenic outcome. Structural analogues of choline and ethanolamine have been shown to be incorporated into phospholipid fractions (20) and to inhibit the uptake of choline into cells (21). In a similar manner, there is evidence that DEA is incorporated into liver phospholipid fractions (22), suggesting that DEA treatment could disrupt choline utilization in cells. Therefore, the purpose of the present work was to determine whether DEA treatment altered the synthesis of PC or the availability of choline in cells. Chinese hamster ovary (CHO) cells
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were used for this work because choline uptake and PC synthesis in these proliferating cells is well characterized (23). MATERIALS AND METHODS Cell culture conditions. CHO-K1 cells (American Type Tissue Culture Collection, CCL-61, Rockville, MD) were plated in 60 mm plastic petri dishes at 3 3 10 5 cells in 3 ml of Ham’s F-12 medium (Life Technologies, Rockville, MD) supplemented with 10% (v/v) sterile fetal bovine serum (Hyclone, Logan, Utah) and 1% penicillin/ streptomycin (Life Technologies, Rockville, MD). The concentration of choline in this medium is 100 mM. In some experiments, choline chloride (Aldrich Chemical Company, Milwaukee, WI) was added to the medium to a final choline concentration of 30 mM. Chemicals and radiochemicals. DEA (99% purity) was obtained from Aldrich Chemical Co. (Milwaukee, WI). 33P-Phosphoric acid and 3 H-choline chloride were from New England Nuclear (Boston, MA), and 14C-DEA was kindly provided by Dr. William Stott (Dow Chemical Co., Midland, MI). Phospholipid synthesis in CHO cells. CHO cells were plated and incubated in complete Ham’s F-12 medium overnight prior to exposure to medium containing DEA. DEA was diluted in Ham’s F-12 medium, pH adjusted to 7.4 6 0.15 and added to complete medium to achieve concentrations of 0, 20, 50, 100, 200, 500 and 1000 mg/ml. Immediately after exposing the cells to DEA, 33P was added (10 mCi/dish) and cells were cultured for an additional 48 hrs. In a separate series of experiments, CHO cells were grown in the presence of DEA and 33P in a medium that was supplemented with 30 mM choline. Cells were cultured for 48 hrs in the cholinesupplemented medium. Additionally, to determine whether DEA was incorporated into phospholipids, CHO cells were grown in complete medium containing 14C-DEA (500 mg/ml; 10 mCi/dish) and cultured for 48 hrs prior to analysis. Cells had not reached confluency by the end of each experiment. Lipid extraction and analysis. Cells were harvested by trypsin digestion after which an aliquot was removed to determine cell number and viability. The remaining cells were pelleted by centrifugation (2000 rpm for 15 min) then resuspended and ruptured by the addition of 100 ml distilled water. Total cellular lipids were extracted according to the method of Bligh and Dyer (24). Individual phospholipids were separated by thin layer chromatography (TLC) on Silica gel G plates (250 mm; Analtech, Newark, DE) which were pre-washed with acetone, dried at 105°F for 30 min and stored desiccated prior to use. Phospholipids were separated with a solvent system (5) of chloroform:methanol:ammonium hydroxide (65:25:4). Incorporation of 33 P or 14C into phospho-lipids was determined with an electronic autoradiographic instrument (InstantImager, Packard, Meridian, CT) and identification of individual phospholipids, particularly PC and phosphatidylethanolamine (PE) was confirmed by TLC separation of authentic standards (Avanti, Alabaster, AL) with molybdenum blue detection (25). Choline uptake studies. CHO cells were plated in 24-well culture plates (2 3 10 5 cells/well) and incubated in 1 ml medium overnight (approximately 50% confluent) prior to the addition of fresh medium containing DEA (0-500 mg/ml) and 3H-choline (5 mCi/ml). After a 10 minute incubation at 37°C, choline uptake was stopped by the addition of 1 ml of ice-cold phosphate-buffered saline (PBS). The medium was removed, the cells were washed three times in PBS, pelleted and then solubilized in 0.1 N NaOH. Cellular protein was determined in an aliquot of the solubilized cells with a micro BCA assay (Pierce, Rockford, IL), and the radioactivity content in the cells was analyzed by liquid scintillation counting (Packard, 2500 TR, Meridian, CT).
FIG. 1. Autoradiographic detection of [ 33P]-phospholipids in control and DEA-treated cells. CHO cells were incubated for 48 hours in the presence of DEA (0-1000 mg/ml) and 33P (10 mCi/dish) after which cellular lipids were extracted and the phospholipids separated by TLC analysis. Panel A shows the typical autoradiographic separation of the [ 33P]-phospholipids. Non-radioactive standards were run to verify R f values for the major lipids, with the location of PE (R f 5 0.46 6 0.01) and PC (R f 5 0.30 6 0.01) indicated. DEA treatment qualitatively changed the TLC profile, with an increase in the band size around PE. As shown in Panel B, DEA treatment caused a concentration-dependent decrease in [ 33P]-PC at concentrations $50 mg/ml. For Panel B, the results represent the mean 6 SE of 6 individual 60 mm dishes of CHO cells.
Data analysis. For the [ 33P]- and [ 14C]-incorporation studies, experiments with at least 6 individual plates were conducted. Choline uptake studies were conducted at least twice with a minimum of three replicates per experiment. Group mean and standard errors were calculated and statistical significance was determined by analysis of variance followed by Fisher’s protected least significant difference test (p , 0.05; Stat View, Abacus Concepts, Berkley, CA).
RESULTS Figure 1 shows the typical autoradiographic detection of the incorporation of 33P-phosphorus into CHO cell phospholipids following TLC separation. The mi-
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FIG. 2. Effect of DEA on [ 3H]-choline uptake in CHO cells. In medium containing 100 mM choline, all DEA concentrations tested (20 –500 mg/ml) inhibited the uptake of choline over a 10 minute interval, with a maximum 95% inhibition observed at the two highest concentrations. The results represent the mean 6 SE of 6 replicate analyses.
gration of authentic, non-radioactive standards was used to verify the identity of these bands (R f 5 0.46 6 0.01 and 0.30 6 0.01 for PE and PC, respectively). The PC fraction accounted for approximately 50% (51.7 6 0.7%) of the total 33P-phosphorus in the phospholipid pool. Over the 48-hr experimental period, DEA had no effect on cell number or total 33P incorporation at concentrations up to 500 mg/ml (results not shown), but it decreased the incorporation of 33P-phosphorus into the PC fraction at concentrations $50 mg/ml. At 1000 mg DEA/ml, a 15% reduction in cell number was observed, and DEA decreased 33P incorporation into PC to only 24% of the total phospholipid fraction. DEA treatment also changed the qualitative TLC profile with an increased band size around PE. To determine whether the reduction in PC synthesis was related to the intracellular availability of choline, the uptake of choline into CHO cells in the presence of DEA was measured. Preliminary experiments indicated that choline uptake from the medium (100 mM choline) into the cells was linear over a 30 minute interval, and Fig. 2 shows that DEA markedly reduced the uptake of choline in a 10-minute experimental period. The uptake of choline was decreased at all concentrations of DEA studied, reaching a maximum 95% inhibition in choline uptake at 250 and 500 mg/ml. To determine whether the effects of DEA on choline uptake and PC synthesis were reversible, CHO cells were exposed to DEA in standard Ham’s F-12 medium or in medium supplemented with 30 mM choline (Fig. 3). When incubated with DEA (500 mg/ml), the utilization of 33P in PC decreased to 27% of total phospholipid,
FIG. 3. Effect of choline supplementation on DEA-dependent changes in PC synthesis. CHO cells were exposed to 500 mg DEA/ml in standard Ham’s F-12 medium or in medium supplemented with 30 mM choline. In contrast to standard medium, the presence of excess choline prevented the decrease in 33P incorporation into PC seen with DEA in standard medium. Results represent the mean 6 SE of 6 individual 60 mm dishes of CHO cells.
whereas DEA had no effect on PC synthesis in cholinesupplemented cells. Figure 4 is a representative autoradiogram of the
FIG. 4. Incorporation of [ 14C]-DEA into CHO cell phospholipids. CHO cells were incubated for 48 hours in the presence of 14C-[DEA] (500 mg/ml; 10 mCi/dish) prior to lipid extraction and TLC analysis. A major band of radioactivity migrated with an R f of 0.5, slightly higher than authentic PE.
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phospholipid fraction of CHO cells following a 48 hour exposure to 14C-DEA (500 mg/ml). Approximately 20% of the radioactive DEA in the cells was extracted in the lipid fraction, and a predominant band migrating with an R f of 0.5 was detected. These results indicated that DEA was incorporated into phospholipids, and although the lipid tail has not been identified, the major DEA-containing phospholipid migrated on the TLC plate with an R f higher than choline and more similar to PE. DISCUSSION In most proliferating cells PC synthesis is closely linked to choline availability (8, 13, 21), and to study this relationship, the typical approach is to reduce or remove choline from the culture medium. In the present studies, DEA treatment reduced PC synthesis in CHO cells, with two mechanisms likely contributing to this effect. First, DEA inhibited choline uptake into CHO cells, an action that can reduce PC synthesis by limiting the availability of choline for the CDP-choline pathway. Second, DEA was incorporated into the phospholipid fraction of CHO cells, suggesting that its own biotransformation utilizes the CDP-choline pathway which may further reduce PC synthesis. Importantly, the addition of excess choline to the culture medium prevented these inhibitory effects, indicating that they are competitive and reversible in nature. Several choline analogues have previously been shown to disrupt choline homeostasis in cell culture. The classical choline uptake inhibitor is hemicholinium-3, which inhibits the high affinity choline transporter in cholinergic synaptosomes (26). However, other compounds such as N-isopropylethanolamine have been shown to inhibit choline uptake into CHO cells, and this inhibition is linked to alterations in cell growth (21). Furthermore, N-isopropylethanolamine is incorporated into CHO cell phospholipids (21), a response also observed with N-diethylethanolamine, N, N-diethylethanolamine and N, N-dimethylethanolamine which are incorporated as CDP-derivatives in isolated hepatocytes and reduce PC synthesis (20). Previous studies have indicated that DEA is incorporated into hepatic phospholipids (22) and that it reduces the incorporation of choline into the PC fraction of rat liver homogenates (27). When considered in combination with the present findings, it appears that biochemically, DEA acts much like other amino alcohols, blocking choline uptake, altering choline utilization, and forming aberrant phospholipids in cellular membranes. Long term administration of DEA is hepatocarcinogenic in mice (19). Consequently, the significance of the present results is that they provide evidence that DEA disrupts choline homeostasis and suggest that the liver
tumors seen following DEA treatment may be caused by a mechanism involving or mimicking choline deficiency. Typically, choline deficiency is produced by removing choline from the diet (4, 16 –18), whereas in the present case, the ability of DEA to directly inhibit choline uptake suggests that it may contribute to an intracellular deficiency of choline, even in the presence of adequate dietary choline. When coupled with the ability of cells to incorporate DEA into phospholipid head groups, it is likely that the intracellular regulation of choline and 1-carbon metabolism is perturbed by chronic DEA treatment, contributing to biochemical changes associated with the carcinogenic outcome of choline deficiency, including aberrant hepatocyte turnover, reduced S-adenosylmethionine levels and alterations in DNA methylation (4, 17, 18). Given the present in vitro results, more work to determine the biochemical and molecular changes observed with DEA treatment in animal models, particularly with respect to altered choline homeostasis, is warranted. REFERENCES 1. Blusztajn, J. K. (1998) Science 281, 794 –795. 2. Zeisel, S. H., daCosta, K-A., Franklin, P. D., Alexander, E. A., Lamont, J. T., Sheard, N. F., and Beiser, A. (1991) FASEB J. 5, 2093–2098. 3. Canty, D. J., and Zeisel, S. H. (1994) Nutrition Rev. 52, 327–339. 4. Zeisel, S. H., and Blusztajn, J. K. (1994) Annu. Rev. Nutr. 14, 269 –296. 5. Glaser, M., Ferguson, K. A., and Vagelos, P. R. (1974) Proc. Natl. Acad. Sci. USA 71, 4072– 4076. 6. White, D. A. (1973) in Form and Function of Phospholipids (Ansell, G. B., Dawson, R. M. C., and Hawthorne, J. N., Eds.), Vol. 3, pp. 441– 482, Elsevier, Amsterdam. 7. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193–214. 8. Zlatkine, P., Leroy, C., Moll, G., and LeGrimellec, C. (1996) Biochem. J. 315, 983–987. 9. Pelech, S., and Vance, D. (1984) Biochim. Biophys. Acta 779, 217–251. 10. Vance, D. E. (1990) Biochem. Cell. Biol. 68, 1151–1165. 11. Eagle, H. (1955) J. Exp. Med. 102, 595– 600. 12. Terce´, F., Brun, H., and Vance, D. E. (1994) J. Lipid Res. 35, 2130 –2142. 13. Albright, C. D., Liu, R., Bethea, T. C., daCosta, K-A., Salganik, R. I., and Zeisel, S. H. (1996). FASEB J. 10, 510 –516. 14. daCosta, K-A., Garner, S. C., Chang, J., and Zeisel, S. H. (1995) Carcinogenesis 16, 327–334. 15. Michael, U. F., Cookson, S. L., Chavez, R., and Pardo, V. (1975) Proc. Soc. Exp. Bio. Med. 150, 672– 676. 16. Newberne, P. M., deCarmargo, J. L. V., and Clark, A. J. (1982) Toxicol. Pathol. 10, 95–106. 17. Rogers, A. E., Nields, H. M., and Newberne, P. M. (1987) Arch. Toxicol. Suppl. 10, 231–243. 18. Lombardi, B., and Smith, M. L. (1996) J. Nutr. Biochem. 5, 2–9. 19. National Toxicology Program (NTP) (1999) Technical Report 478. US Department of Health and Human Services, Public
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Health Service, National Institutes of Health, Bethesda, MD (in press). Akesson, B. (1977) Biochem. J. 168, 401– 408. Borman, L. S. (1982) In Vitro 18, 129 –140. Mathews, J. M., Garner, C. E., and Matthews, H. B. (1995) Chem. Res. Toxicol. 8, 625– 633. Raetz, C. R. H. (1982) in Phospholipids, New Comprehensive Biochemistry (Hawthorne, J. N., and Ansell, G. B., Eds.), Vol. 4, pp. 435– 477, Elsevier, Amsterdam.
24. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911–917. 25. Dittmer, J. C., and Lester, R. L. (1964) J. Lipid Res. 5, 126 –127. 26. Leftkowitz, R. J., Hoffman, B. B., and Taylor, P. (1990) in Goodman and Gilman’s The Pharmacological Basis of Therapeutics (Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P., Eds.), 8th ed., pp. 113–114, Pergamon Press, New York. 27. Barbee, S. J., and Hartung, R. (1979) Toxicol. Appl. Pharmacol. 47, 421– 430.
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Diethanolamine Inhibits Choline Uptake and Phosphatidylcholine Synthesis in Chinese Hamster Ovary Cells Lois D. Lehman-McKeeman 1 and Elizabeth A. Gamsky Human and Environmental Safety Division, Procter and Gamble Co., P.O. Box 538707, Cincinnati, Ohio 45253
Received July 26, 1999
Diethanolamine (DEA), an alkanolamine used widely in industry, is hepatocarcinogenic in mice. The goal of this work was to determine whether DEA altered choline homeostasis in cultured cells, so as to ascertain whether the liver tumor response may be related to choline deficiency. CHO cells were cultured in Ham’s F-12 medium containing DEA (0-1000 mg/ml) and [ 33P]-phosphorus was used to label phospholipid pools. After 48 hours incubation, lipids were extracted and [ 33P]-labeled phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) accounted for 51 6 0.7% of the total lipid 33P incorporation. DEA had no effect on cell number or total phospholipid biosynthesis, but it significantly decreased the incorporation of 33P into PC at concentrations >50 mg/ml. DEA (>20 mg/ml) also inhibited the uptake of [ 3H]-choline into CHO cells, with 95% inhibition observed at 250 mg/ml. To determine whether supplemental choline prevented PC synthesis inhibition by DEA, CHO cells were cultured with or without excess choline (30 mM) and DEA (500 mg/ml). DEA reduced PC synthesis to 27 6 3% of total phospholipids, but had no effect on PC synthesis in choline-supplemented cells. When [ 14C]-DEA was incubated with CHO cells, it was also incorporated into the phospholipid fraction. Collectively, these results indicate that DEA reversibly inhibits PC synthesis by blocking choline uptake and competing for utilization in the CDP-choline pathway in CHO cells. © 1999 Academic Press
Choline is an endogenous compound and dietary component that is essential for the normal function of all cells (1– 4). This quaternary amine is present in tissues primarily as phosphatidylcholine (PC), which accounts for about one half of the total phospholipid 1 Corresponding author. Fax: (513) 627-1908. E-mail: lehman [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
content of mammalian cells (5, 6). Other important choline-containing metabolites include acetylcholine, platelet-activating factor, choline plasmalogens, phosphocholine and betaine (4). In most cells, choline uptake is the first step in the cytidyl diphosphate (CDP)-choline pathway for the biosynthesis of choline-containing phospholipids (7, 8). Once inside the cell, choline is phosphorylated by choline kinase, after which phosphocholine is converted to CDP-choline by CTP-cholinephosphate cytidyltransferase, the regulated, rate-limiting step in PC synthesis (9, 10). CDP-choline in combination with diacylglycerol forms PC and cytidine monophosphate. It is generally recognized that choline is essential for cells because intentional deprivation of choline from cell culture medium disrupts cell growth and division (5, 11–13), and dietary choline deficiency alters hepatic (4, 14) and renal (4, 15) function. Moreover, choline deficiency is the only single-nutrient deficiency that causes spontaneous carcinogenesis in rodents (4, 16 –18). Diethanolamine (DEA), an alkanolamine structurally similar to ethanolamine, is widely used in industry and its fatty acid condensates are present in many household products. Recently, it has been shown that with lifetime exposure, DEA causes liver tumors in mice (19). However, DEA showed no evidence of DNA reactivity, suggesting that a secondary, non-genotoxic mechanism may be involved in the hepatocarcinogenic outcome. Structural analogues of choline and ethanolamine have been shown to be incorporated into phospholipid fractions (20) and to inhibit the uptake of choline into cells (21). In a similar manner, there is evidence that DEA is incorporated into liver phospholipid fractions (22), suggesting that DEA treatment could disrupt choline utilization in cells. Therefore, the purpose of the present work was to determine whether DEA treatment altered the synthesis of PC or the availability of choline in cells. Chinese hamster ovary (CHO) cells
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were used for this work because choline uptake and PC synthesis in these proliferating cells is well characterized (23). MATERIALS AND METHODS Cell culture conditions. CHO-K1 cells (American Type Tissue Culture Collection, CCL-61, Rockville, MD) were plated in 60 mm plastic petri dishes at 3 3 10 5 cells in 3 ml of Ham’s F-12 medium (Life Technologies, Rockville, MD) supplemented with 10% (v/v) sterile fetal bovine serum (Hyclone, Logan, Utah) and 1% penicillin/ streptomycin (Life Technologies, Rockville, MD). The concentration of choline in this medium is 100 mM. In some experiments, choline chloride (Aldrich Chemical Company, Milwaukee, WI) was added to the medium to a final choline concentration of 30 mM. Chemicals and radiochemicals. DEA (99% purity) was obtained from Aldrich Chemical Co. (Milwaukee, WI). 33P-Phosphoric acid and 3 H-choline chloride were from New England Nuclear (Boston, MA), and 14C-DEA was kindly provided by Dr. William Stott (Dow Chemical Co., Midland, MI). Phospholipid synthesis in CHO cells. CHO cells were plated and incubated in complete Ham’s F-12 medium overnight prior to exposure to medium containing DEA. DEA was diluted in Ham’s F-12 medium, pH adjusted to 7.4 6 0.15 and added to complete medium to achieve concentrations of 0, 20, 50, 100, 200, 500 and 1000 mg/ml. Immediately after exposing the cells to DEA, 33P was added (10 mCi/dish) and cells were cultured for an additional 48 hrs. In a separate series of experiments, CHO cells were grown in the presence of DEA and 33P in a medium that was supplemented with 30 mM choline. Cells were cultured for 48 hrs in the cholinesupplemented medium. Additionally, to determine whether DEA was incorporated into phospholipids, CHO cells were grown in complete medium containing 14C-DEA (500 mg/ml; 10 mCi/dish) and cultured for 48 hrs prior to analysis. Cells had not reached confluency by the end of each experiment. Lipid extraction and analysis. Cells were harvested by trypsin digestion after which an aliquot was removed to determine cell number and viability. The remaining cells were pelleted by centrifugation (2000 rpm for 15 min) then resuspended and ruptured by the addition of 100 ml distilled water. Total cellular lipids were extracted according to the method of Bligh and Dyer (24). Individual phospholipids were separated by thin layer chromatography (TLC) on Silica gel G plates (250 mm; Analtech, Newark, DE) which were pre-washed with acetone, dried at 105°F for 30 min and stored desiccated prior to use. Phospholipids were separated with a solvent system (5) of chloroform:methanol:ammonium hydroxide (65:25:4). Incorporation of 33 P or 14C into phospho-lipids was determined with an electronic autoradiographic instrument (InstantImager, Packard, Meridian, CT) and identification of individual phospholipids, particularly PC and phosphatidylethanolamine (PE) was confirmed by TLC separation of authentic standards (Avanti, Alabaster, AL) with molybdenum blue detection (25). Choline uptake studies. CHO cells were plated in 24-well culture plates (2 3 10 5 cells/well) and incubated in 1 ml medium overnight (approximately 50% confluent) prior to the addition of fresh medium containing DEA (0-500 mg/ml) and 3H-choline (5 mCi/ml). After a 10 minute incubation at 37°C, choline uptake was stopped by the addition of 1 ml of ice-cold phosphate-buffered saline (PBS). The medium was removed, the cells were washed three times in PBS, pelleted and then solubilized in 0.1 N NaOH. Cellular protein was determined in an aliquot of the solubilized cells with a micro BCA assay (Pierce, Rockford, IL), and the radioactivity content in the cells was analyzed by liquid scintillation counting (Packard, 2500 TR, Meridian, CT).
FIG. 1. Autoradiographic detection of [ 33P]-phospholipids in control and DEA-treated cells. CHO cells were incubated for 48 hours in the presence of DEA (0-1000 mg/ml) and 33P (10 mCi/dish) after which cellular lipids were extracted and the phospholipids separated by TLC analysis. Panel A shows the typical autoradiographic separation of the [ 33P]-phospholipids. Non-radioactive standards were run to verify R f values for the major lipids, with the location of PE (R f 5 0.46 6 0.01) and PC (R f 5 0.30 6 0.01) indicated. DEA treatment qualitatively changed the TLC profile, with an increase in the band size around PE. As shown in Panel B, DEA treatment caused a concentration-dependent decrease in [ 33P]-PC at concentrations $50 mg/ml. For Panel B, the results represent the mean 6 SE of 6 individual 60 mm dishes of CHO cells.
Data analysis. For the [ 33P]- and [ 14C]-incorporation studies, experiments with at least 6 individual plates were conducted. Choline uptake studies were conducted at least twice with a minimum of three replicates per experiment. Group mean and standard errors were calculated and statistical significance was determined by analysis of variance followed by Fisher’s protected least significant difference test (p , 0.05; Stat View, Abacus Concepts, Berkley, CA).
RESULTS Figure 1 shows the typical autoradiographic detection of the incorporation of 33P-phosphorus into CHO cell phospholipids following TLC separation. The mi-
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FIG. 2. Effect of DEA on [ 3H]-choline uptake in CHO cells. In medium containing 100 mM choline, all DEA concentrations tested (20 –500 mg/ml) inhibited the uptake of choline over a 10 minute interval, with a maximum 95% inhibition observed at the two highest concentrations. The results represent the mean 6 SE of 6 replicate analyses.
gration of authentic, non-radioactive standards was used to verify the identity of these bands (R f 5 0.46 6 0.01 and 0.30 6 0.01 for PE and PC, respectively). The PC fraction accounted for approximately 50% (51.7 6 0.7%) of the total 33P-phosphorus in the phospholipid pool. Over the 48-hr experimental period, DEA had no effect on cell number or total 33P incorporation at concentrations up to 500 mg/ml (results not shown), but it decreased the incorporation of 33P-phosphorus into the PC fraction at concentrations $50 mg/ml. At 1000 mg DEA/ml, a 15% reduction in cell number was observed, and DEA decreased 33P incorporation into PC to only 24% of the total phospholipid fraction. DEA treatment also changed the qualitative TLC profile with an increased band size around PE. To determine whether the reduction in PC synthesis was related to the intracellular availability of choline, the uptake of choline into CHO cells in the presence of DEA was measured. Preliminary experiments indicated that choline uptake from the medium (100 mM choline) into the cells was linear over a 30 minute interval, and Fig. 2 shows that DEA markedly reduced the uptake of choline in a 10-minute experimental period. The uptake of choline was decreased at all concentrations of DEA studied, reaching a maximum 95% inhibition in choline uptake at 250 and 500 mg/ml. To determine whether the effects of DEA on choline uptake and PC synthesis were reversible, CHO cells were exposed to DEA in standard Ham’s F-12 medium or in medium supplemented with 30 mM choline (Fig. 3). When incubated with DEA (500 mg/ml), the utilization of 33P in PC decreased to 27% of total phospholipid,
FIG. 3. Effect of choline supplementation on DEA-dependent changes in PC synthesis. CHO cells were exposed to 500 mg DEA/ml in standard Ham’s F-12 medium or in medium supplemented with 30 mM choline. In contrast to standard medium, the presence of excess choline prevented the decrease in 33P incorporation into PC seen with DEA in standard medium. Results represent the mean 6 SE of 6 individual 60 mm dishes of CHO cells.
whereas DEA had no effect on PC synthesis in cholinesupplemented cells. Figure 4 is a representative autoradiogram of the
FIG. 4. Incorporation of [ 14C]-DEA into CHO cell phospholipids. CHO cells were incubated for 48 hours in the presence of 14C-[DEA] (500 mg/ml; 10 mCi/dish) prior to lipid extraction and TLC analysis. A major band of radioactivity migrated with an R f of 0.5, slightly higher than authentic PE.
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phospholipid fraction of CHO cells following a 48 hour exposure to 14C-DEA (500 mg/ml). Approximately 20% of the radioactive DEA in the cells was extracted in the lipid fraction, and a predominant band migrating with an R f of 0.5 was detected. These results indicated that DEA was incorporated into phospholipids, and although the lipid tail has not been identified, the major DEA-containing phospholipid migrated on the TLC plate with an R f higher than choline and more similar to PE. DISCUSSION In most proliferating cells PC synthesis is closely linked to choline availability (8, 13, 21), and to study this relationship, the typical approach is to reduce or remove choline from the culture medium. In the present studies, DEA treatment reduced PC synthesis in CHO cells, with two mechanisms likely contributing to this effect. First, DEA inhibited choline uptake into CHO cells, an action that can reduce PC synthesis by limiting the availability of choline for the CDP-choline pathway. Second, DEA was incorporated into the phospholipid fraction of CHO cells, suggesting that its own biotransformation utilizes the CDP-choline pathway which may further reduce PC synthesis. Importantly, the addition of excess choline to the culture medium prevented these inhibitory effects, indicating that they are competitive and reversible in nature. Several choline analogues have previously been shown to disrupt choline homeostasis in cell culture. The classical choline uptake inhibitor is hemicholinium-3, which inhibits the high affinity choline transporter in cholinergic synaptosomes (26). However, other compounds such as N-isopropylethanolamine have been shown to inhibit choline uptake into CHO cells, and this inhibition is linked to alterations in cell growth (21). Furthermore, N-isopropylethanolamine is incorporated into CHO cell phospholipids (21), a response also observed with N-diethylethanolamine, N, N-diethylethanolamine and N, N-dimethylethanolamine which are incorporated as CDP-derivatives in isolated hepatocytes and reduce PC synthesis (20). Previous studies have indicated that DEA is incorporated into hepatic phospholipids (22) and that it reduces the incorporation of choline into the PC fraction of rat liver homogenates (27). When considered in combination with the present findings, it appears that biochemically, DEA acts much like other amino alcohols, blocking choline uptake, altering choline utilization, and forming aberrant phospholipids in cellular membranes. Long term administration of DEA is hepatocarcinogenic in mice (19). Consequently, the significance of the present results is that they provide evidence that DEA disrupts choline homeostasis and suggest that the liver
tumors seen following DEA treatment may be caused by a mechanism involving or mimicking choline deficiency. Typically, choline deficiency is produced by removing choline from the diet (4, 16 –18), whereas in the present case, the ability of DEA to directly inhibit choline uptake suggests that it may contribute to an intracellular deficiency of choline, even in the presence of adequate dietary choline. When coupled with the ability of cells to incorporate DEA into phospholipid head groups, it is likely that the intracellular regulation of choline and 1-carbon metabolism is perturbed by chronic DEA treatment, contributing to biochemical changes associated with the carcinogenic outcome of choline deficiency, including aberrant hepatocyte turnover, reduced S-adenosylmethionine levels and alterations in DNA methylation (4, 17, 18). Given the present in vitro results, more work to determine the biochemical and molecular changes observed with DEA treatment in animal models, particularly with respect to altered choline homeostasis, is warranted. REFERENCES 1. Blusztajn, J. K. (1998) Science 281, 794 –795. 2. Zeisel, S. H., daCosta, K-A., Franklin, P. D., Alexander, E. A., Lamont, J. T., Sheard, N. F., and Beiser, A. (1991) FASEB J. 5, 2093–2098. 3. Canty, D. J., and Zeisel, S. H. (1994) Nutrition Rev. 52, 327–339. 4. Zeisel, S. H., and Blusztajn, J. K. (1994) Annu. Rev. Nutr. 14, 269 –296. 5. Glaser, M., Ferguson, K. A., and Vagelos, P. R. (1974) Proc. Natl. Acad. Sci. USA 71, 4072– 4076. 6. White, D. A. (1973) in Form and Function of Phospholipids (Ansell, G. B., Dawson, R. M. C., and Hawthorne, J. N., Eds.), Vol. 3, pp. 441– 482, Elsevier, Amsterdam. 7. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193–214. 8. Zlatkine, P., Leroy, C., Moll, G., and LeGrimellec, C. (1996) Biochem. J. 315, 983–987. 9. Pelech, S., and Vance, D. (1984) Biochim. Biophys. Acta 779, 217–251. 10. Vance, D. E. (1990) Biochem. Cell. Biol. 68, 1151–1165. 11. Eagle, H. (1955) J. Exp. Med. 102, 595– 600. 12. Terce´, F., Brun, H., and Vance, D. E. (1994) J. Lipid Res. 35, 2130 –2142. 13. Albright, C. D., Liu, R., Bethea, T. C., daCosta, K-A., Salganik, R. I., and Zeisel, S. H. (1996). FASEB J. 10, 510 –516. 14. daCosta, K-A., Garner, S. C., Chang, J., and Zeisel, S. H. (1995) Carcinogenesis 16, 327–334. 15. Michael, U. F., Cookson, S. L., Chavez, R., and Pardo, V. (1975) Proc. Soc. Exp. Bio. Med. 150, 672– 676. 16. Newberne, P. M., deCarmargo, J. L. V., and Clark, A. J. (1982) Toxicol. Pathol. 10, 95–106. 17. Rogers, A. E., Nields, H. M., and Newberne, P. M. (1987) Arch. Toxicol. Suppl. 10, 231–243. 18. Lombardi, B., and Smith, M. L. (1996) J. Nutr. Biochem. 5, 2–9. 19. National Toxicology Program (NTP) (1999) Technical Report 478. US Department of Health and Human Services, Public
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