Choline Supplementation Inhibits Diethanolamine-Induced Morphological Transformation in Syrian Hamster Embryo Cells: Evidence for a Carcinogenic Mechanism

Lois D. Lehman-McKeeman1 and Elizabeth A. Gamsky

Human and Environmental Safety Division, Miami Valley Laboratories, Procter and Gamble Company, Cincinnati, Ohio 45253

Received November 24, 1999; accepted February 2, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DEA, an amino alcohol, and its fatty acid condensates are widely used in commerce. DEA is hepatocarcinogenic in mice, but shows no evidence of mutagenicity or clastogenicity in a standard testing battery. However, it increased the number of morphologically transformed colonies in the Syrian hamster embryo (SHE) cell morphologic transformation assay. The goal of this work was to test the hypothesis that DEA treatment causes morphologic transformation by a mechanism involving altered cellular choline homeostasis. As a first step, the ability of DEA to disrupt the uptake and intracellular utilization of choline was characterized. SHE cells were cultured in medium containing DEA (500 µg/ml), and 33P-phosphorus or 14C-choline was used to label phospholipid pools. After 48 h, SHE cells were harvested, lipids were extracted, and radioactive phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) was the major phospholipid, accounting for 43 ± 1% of total phospholipid synthesis. However, with DEA treatment, PC was reduced to 14 ± 2% of total radioactive phospholipids. DEA inhibited choline uptake into SHE cells at concentrations >= 50 µg /ml, reaching a maximum 80% inhibition at 250–500 µg/ml. The concentration dependence of the inhibition of PC synthesis by DEA (0, 10, 50, 100, 250, and 500 µg/ml) was determined in SHE cells cultured over a 7-day period under the conditions of the transformation assay and in the presence or absence of excess choline (30 mM). DEA treatment decreased PC synthesis at concentrations >= 100 µg/ml, reaching a maximum 60% reduction at 500 µg/ml. However, PC synthesis was unaffected when DEA-treated cells were cultured with excess choline. Under 7-day culture conditions, 14C-DEA was incorporated into SHE lipids, and this perturbation was also inhibited by choline supplementation. Finally, DEA (10–500 µg/ml) transformed SHE cells in a concentration-dependent manner, whereas with choline supplementation, no morphologic transformation was observed. Thus, DEA disrupts intracellular choline homeostasis by inhibiting choline uptake and altering phospholipid synthesis. However, excess choline blocks these biochemical effects and inhibits cell transformation, suggesting a relationship between the two responses. Overall, the results provide a plausible mechanism to explain the morphologic transformation observed with DEA and suggest that the carcinogenic effects of DEA may be caused by intracellular choline deficiency.

Key Words: carcinogenesis; cell transformation; choline deficiency; diethanolamine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diethanolamine (DEA) is an alkanolamine that is widely used in industry, and its fatty acid condensates are present in many consumer products. With lifetime dermal exposure, DEA increased the incidence and multiplicity of liver tumors in mice, but no carcinogenic activity was observed in rats (NTP, 1999). However, there was no evidence of DNA reactivity (Knaak et al., 1997Go; NTP, 1999), suggesting that secondary, nongenotoxic mechanisms are likely contributing to the hepatocarcinogenic response.

DEA is structurally similar to choline and ethanolamine, important endogenous precursors in the synthesis of phospholipids essential for normal membrane structure and function (Canty and Zeisel, 1994Go; Pelech and Vance, 1984Go; Zeisel and Blusztajn, 1994Go). Choline was recognized many years ago as vital for maintaining cell viability (Eagle, 1955Go). It is found in tissues primarily as phosphatidylcholine (PC), which accounts for about one-half of the total phospholipid content of all mammalian cells (Glaser et al., 1974Go). In addition to membrane integrity, choline and choline-containing metabolites such as acetylcholine, platelet activating factor, and sphingomyelin are important for neurotransmission and cell-signaling mechanisms (Canty and Zeisel, 1994Go; Spiegel and Merrill, 1996Go; Zeisel and Blusztajn, 1994Go). Moreover, choline can be oxidized to betaine, an essential intracellular osmolyte (Beck et al., 1998Go; Garcia-Perez and Burg, 1991Go) and methyl group donor (Finkelstein et al., 1982Go). The physiologic significance of choline is substantiated by the fact that intentional deprivation of choline disrupts cell growth and division (Albright et al., 1996Go; Terce et al., 1994Go; Yen et al., 1999Go) and dietary choline deficiency alters hepatic (Lombardi, 1971Go; Zeisel and Blusztajn, 1994Go) and renal (Kratzing et al., 1972Go; Michael et al., 1975Go) function. Moreover, dietary choline deprivation is the only single nutrient deficiency that causes spontaneous carcinogenesis in rodents (Ghoshal and Farber, 1984Go; Mikol et al., 1983Go; Newberne et al., 1982Go; Newberne and Rogers, 1986Go; Zeisel, 1996Go).

The major pathway by which PC is synthesized is the cytidyl diphosphate (CDP)-choline pathway (Kennedy and Weiss, 1956Go; Kennedy, 1989Go; Kent, 1995Go; Vance, 1990Go). In some tissues, an alternate pathway involving the sequential methylation of phosphatidylethanolamine (PE) to PC serves as a secondary source of this essential phospholipid (Vance and Ridgeway, 1988). Disposition studies have demonstrated the presence of DEA in phospholipid head groups, suggesting that this unnatural alkanolamine can be utilized in the cellular pathways of phospholipid biosynthesis (Mathews et al., 1995Go; 1997Go). Furthermore, in Chinese hamster ovary (CHO) cells, DEA treatment selectively reduced PC synthesis, with two inter-related mechanisms accounting for this response. Specifically, DEA inhibited the uptake of choline into the cells, thereby limiting the availability of the essential precursor, and DEA was incorporated into phospholipids, again showing that its biotransformation utilized normal cellular phospholipid biosynthetic pathways (Lehman-McKeeman and Gamsky, 1999Go).

As previously noted, although DEA is carcinogenic, genetic toxicology data for DEA are uniformly negative. In direct contrast to the genetic toxicology data, DEA was positive in the Syrian hamster embryo (SHE) cell transformation assay, particularly with a 7-day exposure period (Kerckaert et al. 1996aGo). The SHE assay exhibits approximately 85% concordance with carcinogenic outcome in rat and mouse models (Leboeuf et al. 1999) and is recognized as a useful tool for predicting carcinogenic potential (Leboeuf et al., 1996; IARC, 1999). Thus, DEA-induced SHE cell transformation appears to be predictive of its carcinogenic potential. However, the ability of DEA to disrupt cellular choline homeostasis suggests that the cell transformation, and ultimately carcinogenicity of DEA, may be mechanistically related to its ability to produce intracellular choline deficiency, a known carcinogenic insult (Zeisel, 1996Go).

The purpose of present studies was to test the hypothesis that DEA treatment causes cell transformation by a mechanism involving the development of choline deficiency. Accordingly, the first objective was to characterize the biochemical effects of DEA in SHE cells, including its ability to disrupt the cellular uptake and utilization of choline. The second objective was to determine whether these biochemical alterations were associated with the cell transformation response. To accomplish this goal, DEA-induced cell transformation was evaluated with SHE cells cultured in normal and choline-supplemented media. Collectively, the biochemical and cellular results provide the foundation for elucidating the molecular mechanisms of DEA-induced carcinogenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and radiochemicals.
DEA (99% purity) was obtained from Aldrich Chemical Co. (Milwaukee, WI). 33P-Phosphoric acid (8810 Ci/mmol), [methyl –14C] choline chloride (54 mCi/mmol) and [methyl-3H] choline chloride (81 Ci/mmol) were from New England Nuclear (Boston, MA). 14C-DEA (universally labeled; hydrochloride salt) was synthesized by New England Nuclear with a specific activity of 46 mCi/mmol and a radiochemical purity of 98%. All other reagents were cell culture grade or better.

General cell culture conditions.
Cryopreserved, primary isolates of SHE cells were obtained from Covance (Vienna, VA), and cells were grown in Dulbecco's Modified Eagle's Medium-Leboeuf's modification (DMEM-L; Quality Biologicals, Gaithersburg, MD) containing 20% (v/v) sterile fetal bovine serum (Hyclone, Logan, Utah) and 4 mM-glutamine (Gibco, Gaithersburg, MD). The major modification in DMEM-L was a reduction in NaHCO3 (Leboeuf et al., 1989Go), and the concentration of choline (as choline chloride) in this medium was 28 µM. Cells were incubated at 37 ± 1° C and 10 ± 0.5% CO2. Under these conditions, the pH of DMEM-L is 6.70.

Effects of DEA on SHE cell phospholipid biosynthesis.
In the first set of experiments, the cryopreserved SHE cells were thawed, grown to 50–90% confluency, then subcultured and plated at a density of 1 x 105 cells in 60-mm plastic petri dishes (3 ml). The cells were incubated overnight prior to exposure to DEA, which was diluted in DMEM-L, pH adjusted to 7.00 ± 0.02 (ambient conditions; pH 6.7 at 10% CO2), and added to complete medium to achieve a final concentration of 0 or 500 µg/ml. Immediately after exposing the cells to DEA, 33P-phosphoric acid or 14C-choline chloride was added (10 µCi/dish) to label the phospholipid pools. Cells were cultured for an additional 48 h before harvesting. Cells had not reached confluency by the end of these experiments. Each experiment was conducted in triplicate, and three separate experiments were performed.

In a second series of experiments, the biosynthesis of phospholipids in DEA-exposed SHE cells was studied under the conditions of the typical 7-day transformation assay (Kerckaert et al., 1996bGo). Briefly, feeder SHE cells (4 x 104 cells), prepared by X-ray irradiation (approximately 5000 rad) to be viable but no longer replicating, were incubated overnight in 60-mm culture dishes after which 2 ml of a second SHE-cell suspension (target cells; 30–50 cells/ml) was added to the X-irradiated SHE cells. The cells were incubated overnight prior to exposure to DEA, which was diluted in DMEM-L, pH adjusted to 7.00 ± 0.02 (ambient conditions; pH 6.7 at 10% CO2), and added to complete medium to achieve a final concentration of 0, 10, 50, 100, 250, or 500 µg/ml. In some experiments, choline chloride was added to the complete medium to a final choline concentration of 30 mM. The level of choline supplementation was selected to be a large molar excess relative to both constitutive choline in the medium (about 1000-times higher) and the DEA added (6–300 times higher) without markedly altering the osmolarity or pH of the medium. For these cultures, the choline-supplemented medium was applied to the cells just prior to adding DEA, and immediately thereafter, 33P-phosphoric was added (10 µCi/dish). Cells were cultured for an additional 7 days before harvesting. Three separate experiments (with two to three replicates per experiment) were completed.

To determine whether DEA was incorporated into SHE-cell phospholipids, a 7-day culture experiment was performed as described above in which cells were exposed to 14C-DEA (500 µg/ml; 50 µCi/dish) in the presence or absence of the choline-supplemented medium (30 mM). During the phospholipid extraction, the amount of DEA in the aqueous and organic fractions of the cells was also determined by liquid scintillation counting (Packard, 2500 TR, Meridian, CT). This experiment was conducted twice with three replicates per experiment.

Lipid extraction and analysis.
Cells were harvested by trypsin digestion, after which an aliquot was removed to determine cell number and viability. In the 48-h mass culture experiments, cells were counted with either a hemocytometer or Coulter counter (Coulter Z1, Miami, FL) and viability was determined by trypan blue exclusion. For the 7-day experiments, total cellular protein (target plus feeder cells) was quantified as described below. Cells were harvested by centrifugation (2000 rpm for 15 min), then resuspended and disrupted by the addition of approximately 100 µl distilled water. Total cellular lipids were extracted according to the method of Bligh and Dyer (1959). Briefly, the disrupted suspension was extracted with chloroform:methanol (1:2), and mixed thoroughly prior to adding a second aliquot of chloroform; the aqueous and organic layers were separated by centrifugation (2000 x g for 5 min). Unless used for other analyses, the aqueous layer was discarded, after which the samples were extracted a second time with chloroform. The combined organic layer was evaporated under a stream of N2 and reconstituted in 25 µl chloroform pending analysis. Individual phospholipids were separated by thin layer chromatography (TLC) on silica gel G plates (250 µm; Analtech, Newark, DE), which were prewashed with acetone, dried at 105° F for 30 min, and stored desiccated prior to use. For this analysis, 5–10 µl of the reconstituted lipid fraction was applied to the TLC plate, and phospholipids were separated with a solvent system consisting of chloroform:methanol:ammonium hydroxide (65:25:4), as described by Glaser et al. (1974). All samples were analyzed in duplicate runs on separate TLC plates. Incorporation of 33P or 14C into phospholipids was determined with an electronic autoradiographic instrument (InstantImager, Packard, Meridian, CT) in which the total radioactivity in each lane, along with the radioactivity in each phospholipid fraction, was quantified. The identification of individual phospholipids, particularly PC and PE, was confirmed by TLC separation of authentic standards (Avanti, Alabaster, AL) with molybdenum blue detection (Dittmer and Lester, 1964Go).

Choline uptake studies.
The uptake of 3H-choline was determined in mass-cultured SHE cells in the absence or presence of DEA. A series of preliminary time-course experiments (1, 2, 5, 10, 20, and 30 min) demonstrated that choline uptake from control medium was linear for 20 min (results not shown). Therefore, for all subsequent experiments, the uptake was quantified over a 10-min interval. SHE cells were subcultured in 24-well culture plates (2 x 105 cells/well) and incubated in 1 ml complete medium overnight (approximately 50% confluent) prior to the addition of fresh medium containing DEA (0, 10, 50, 100, 250, or 500 µg/ml) and 3H-choline (5 µCi/ml). After a 10-min 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, and then solubilized in 0.1N NaOH. The radioactivity content in the washed and solubilized cells was analyzed by liquid scintillation counting (Packard, 2500 TR, Meridian, CT), and a separate aliquot was used for protein analyses. Uptake studies were conducted in two separate experiments with a minimum of three replicates per experiment.

SHE-cell transformation assay.
The potential for DEA to increase the frequency of morphologic transformation in SHE cells was determined in a 7-day continuous exposure protocol (Kerckaert et al., 1996bGo). Preliminary cytotoxicity experiments indicated that plating efficiency was reduced by 25–30% at 500 µg DEA/ml in a 7-day assay, and that the addition of 30 mM choline to the DMEM-L increased the osmolarity of the medium to approximately 330 mOsm/kg, but had no effect on plating efficiency. Two independent assays were conducted. In the first assay, the transforming potential of DEA was tested at 0, 10, 50, 100, 250, and 500 µg/ml, whereas the second assay was performed using choline-supplemented medium at the same concentrations of DEA. Benzo[a]pyrene (5 µg/ml) served as a positive control. A total of 40 culture dishes (60 mm) were prepared for each concentration of DEA tested, and the total colonies analyzed at each concentration exceeded 1000. At the end of the 7-day exposure, the culture medium was removed and the colonies were fixed in methanol and stained with Giemsa. The total number of colonies was counted to determine the relative plating efficiency, after which the total number of transformed colonies was determined. Morphologic evaluations were performed with no knowledge of treatment group.

Other assays.
In conducting the 7-day phospholipid biosynthesis and choline uptake studies, cellular protein was measured. For this work, an aliquot of the trypsinized cells was solubilized in 0.1N NaOH and protein was determined with a micro BCA assay (Pierce, Rockford, IL).

Data analysis.
For the 33P- and 14C-incorporation and 3H-choline uptake studies, group mean and standard errors were determined across experiments. 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).

For the SHE cell transformation assay, the morphologic transformation frequency (as a percent of total colonies) was calculated, after which the statistical significance between each concentration tested and the solvent control was evaluated with a one-sided Fisher's Exact test (p < 0.05). In addition, the transformation data were evaluated with an unstratified binomial exact permutation trend test for a positive dose response (p < 0.05). A test chemical is considered to be positive in the SHE-cell transformation assay if it causes a statistically significant increase in the morphologic transformation frequency in at least two test concentrations, or if the transformation frequency is statistically significant at one concentration and the dose-response trend test is positive (Kerckaert et al., 1996bGo).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In these studies, the incorporation of 33P-phosphorus into cellular lipid pools was used as a measure of phospholipid biosynthesis. Total incorporation of the radioactive precursor over a 48-h or 7-day interval most accurately represents the net balance between phospholipid synthesis and catabolism. However, newly synthesized phospholipids are necessary to sustain the clonal growth of cells (Terce et al., 1994Go), making it very likely that phospholipid synthesis predominates over catabolism in the SHE cells.

The autoradiographic TLC separation of SHE-cell phospholipids labeled with 33P-phosphorus or 14C-choline is shown in Figure 1Go. The migration of authentic, nonradioactive standards was used to verify the identity of the bands, with the migration of PC and PE indicated. With these methods, the major phospholipids were readily detected in untreated SHE cells cultured over a 48 hr-period in the presence of the radioactive precursors (Fig. 1Go, lanes 1 and 2 and 5 and 6 for 33P-phosphorus and 14C-choline, respectively). PC is the major phospholipid in the cells, and the band that migrated with authentic PC represented 43 ± 1% (n = 9) of total 33P-labeled lipids.



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FIG. 1. Autoradiographic detection of 33P- or 14C-choline containing phospholipids in control (Lanes 1, 2, 5, and 6) and DEA-treated (Lanes 3, 4, 7, and 8) cells. SHE cells were incubated for 48 h in the presence of DEA (0 or 500 µg/ml), after which cellular lipids were extracted and the phospholipids were separated by TLC. Nonradioactive standards were run to verify the migration of the major lipids (PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomyelin; LyPC, lysophosphatidylcholine). DEA treatment decreased the incorporation of both radioactive precursors into the PC fraction, and with 33P-incorporation, a qualitative change in the TLC profile, particularly in the band size around PE, was noted.

 
Figure 1Go also shows the effects the DEA treatment on 33P-phosphorus and 14C-choline incorporation into the phospholipid fraction of SHE cells cultured over a 48-h treatment period. At 500 µg/ml, DEA decreased cell number by about 10% but had no effect on the total cellular 33P-incorporation (results not shown). However, DEA treatment decreased the incorporation of 33P into the PC fraction to 14 ± 2% of the total radioactivity in phospholipids (Fig. 1Go, lanes 3 and 4). In addition, DEA treatment qualitatively altered the TLC profile, as evidenced by a large band that extended beyond the migration of authentic PE. The phospholipid(s) in this fraction have not yet been determined.

To specifically evaluate the effects of DEA on choline-containing phospholipids, the incorporation of 14C-choline into SHE cell lipids was measured. DEA treatment (500 µg/ml; Fig. 1Go, lanes 7 and 8) reduced the radioactivity in the PC fraction from 467 ± 17 dpm to 149 ± 15 dpm (n = 4), or approximately a 70% reduction relative to control. Synthesis of other choline-containing phospholipids, including those migrating with an Rf consistent with lysophophosphatidylcholine and sphingomyelin, was also reduced relative to untreated cells.

Given that DEA treatment reduced PC synthesis in SHE cells, its ability to also alter the intracellular availability of choline was determined. Choline uptake was quantified in the presence and absence of DEA, over a time interval (10 min) during which uptake was linear. Figure 2Go demonstrates that at concentrations >= 50 µg/ml, DEA significantly reduced choline uptake, reaching a maximum of about 85% inhibition relative to untreated cells.



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FIG. 2. Effect of DEA treatment on 3H-choline uptake in SHE cells. When cultured in DMEM-L (28 µM choline), concentrations of DEA at 50 µg/ml and higher significantly reduced choline uptake measured over a 10-min interval. The results represent the mean ± SE of six replicates and (*) denotes p < 0.05.

 
Figure 3Go is a representative autoradiogram showing the incorporation of 33P-phosphorus and 14C-DEA into SHE-cell phospholipids in cells cultured for a 7-day period under the standard experimental conditions used for the transformation assay. In these experiments, the effect of choline supplementation (30 mM) on phospholipid synthesis was also determined. When 33P-phosphorus was used to label all phospholipids, DEA treatment (500 µg/ml) again reduced PC synthesis by about 60%, whereas choline supplementation prevented this reduction. In addition, when cultured for 7 days in the presence of 14C-DEA, approximately 12% (11.8 ± 0.1%; n = 6) of the intracellular DEA was present in the lipid fraction, and SHE cells showed a marked accumulation of DEA into aberrant phospholipids which migrated with an Rf similar to PE. When cells were cultured in the presence of excess choline, no DEA incorporation into phospholipids was observed.



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FIG. 3. Incorporation of 33P or 14C-DEA (500 µg/ml; 50 µCi/dish) into SHE cell phospholipids in the presence or absence of excess choline. SHE cells were cultured for 7 days in the presence of DEA and the radioactive precursor as noted in normal DMEM-L or medium supplemented with 30 mM choline. The (–) or (+) symbols denote the absence or presence of either DEA or excess choline. DEA treatment decreased 33P-incorporation into PC, but this was prevented in the presence of excess choline. Likewise, 14C-DEA was detected in a lipid fraction migrating in a manner similar to PE, whereas no incorporation was observed in the presence of excess choline.

 
The concentration dependence of the inhibition of PC synthesis in DEA-treated SHE cells cultured over a 7-day period (transformation assay conditions) in the presence or absence of excess choline is shown in Figure 4Go. In standard medium, DEA reduced PC synthesis at concentrations of 100 µg/ml and higher. At 500 µg/ml, less than 20% of the total lipid fraction represented PC, again about a 60% reduction in the total PC content relative to control. In direct contrast, DEA had no effect on PC synthesis in the cells supplemented with choline.



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FIG. 4. Effect of choline supplementation on the concentration dependence of DEA-induced changes in PC synthesis in SHE cells. SHE cells were cultured for 7 days in the presence of DEA (0–500 µg/ml) and 33P-incorporation into the PC fraction was determined by autoradiographic TLC analysis. In standard DMEM-L, DEA decreased PC synthesis, whereas choline supplementation (+ choline; 30 mM) prevented the decrease in 33P-incorporation at all concentrations. The results represent the mean ± SE of eight individual 60-mm dishes and (*) denotes p < 0.05.

 
To determine whether DEA-induced alterations in choline availability and PC synthesis in SHE cells were involved in morphologic transformation, 7-day cell transformation assays were conducted under normal conditions and in medium supplemented with 30 mM choline. The results of these assays (Fig. 5Go) confirmed that under standard assay conditions, DEA gave a positive response, as statistical significance was observed with one concentration along with a positive trend test (Kerckaert et al., 1996bGo). However, in a manner consistent with its ability to prevent the DEA-induced changes in PC synthesis, no morphologic transformation was observed when DEA-treated SHE cells were cultured in choline-supplemented medium. In direct contrast to the results obtained with DEA, choline supplementation had no effect on the morphologic transformation observed with benzo[a]pyrene (results not shown).



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FIG. 5. Effect of choline supplementation on DEA-induced morphologic transformation in SHE cells. Under standard assay conditions (medium), DEA was considered positive because it increased morphologic transformation at 500 µg/ml and exhibited a positive dose response (p < 0.05). In the presence of 30 mM choline (+ choline), no increase in morphologic transformation was observed. Choline supplementation had no effect on the morphologic transformation frequency observed for the positive control, benzo[a]pyrene (5 µg/ml).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present experiments demonstrate that DEA alters choline homeostasis in SHE cells, an action that may be associated with, if not causally related to, DEA-induced morphologic transformation in SHE cells. There are three important features of effects of DEA on choline homeostasis in SHE cells:

Choline transport into cells occurs by three distinct mechanisms, depending on cell type. Cholinergic neurons have a high-affinity, Na-dependent transporter mechanism (Kt < 5 µM; the concentration of choline at half-maximal transport) that is tightly coupled to the synthesis of acetylcholine and which is selectively inhibited by hemicholinium-3 (Ishidate, 1989Go). Noncholinergic cells possess a lower-affinity system (Kt > 30 µM) that is relatively Na-independent and insensitive to hemicholinium-3, and finally, at very high concentrations, choline can enter cells by simple diffusion (Ishidate, 1989Go). The concentration of choline in the DMEM-L medium used in the present studies is 28 µM, indicating that the low-affinity carrier-mediated transporter is primarily responsible for choline uptake in SHE cells, and that this transport system is inhibited by DEA. Thus, the reduction in the overall synthesis of PC and other choline-containing phospholipids in SHE cells exposed to DEA is directly related to the reduction in the intracellular availability of choline. Other choline analogues such as N-isopropylethanolamine have been shown to inhibit choline uptake, and this inhibition resulted in concentration-dependent alterations in cell growth (Borman, 1982Go).

The CDP-choline pathway is a multistep cycle in which intracellular choline is rapidly phosphorylated by choline kinase and then converted to CDP-choline by CTP:phosphocholine cytidyltransferase, the rate-limiting step in the pathway. CDP-choline, in combination with diacylglycerol, forms PC and cytidine monophosphate, a reaction catalyzed by CDP-choline:1,2-diacylglycerol choline-phosphotransferase (Kennedy and Weiss, 1956Go; Kent, 1995Go; Vance, 1990Go). The detection of DEA in SHE cell phospholipids indicates that this incorporation occurred, at least in part, by the formation of CDP-derivatives of DEA. Consequently, the formation of PC, an ongoing cellular process, is likely to be reduced or inhibited when cells form aberrant, DEA-containing phospholipids. This effect is not without precedent, as several choline analogues, including N, N-diethylethanolamine and N, N-dimethylethanolamine, have been shown to selectively inhibit PC synthesis by competing for the CDP-choline pathway (Akesson, 1977Go). Furthermore, the present data with cultured cells are consistent with observations from animal studies indicating that DEA can be incorporated into cellular lipid fraction (Mathews et al., 1995Go) and that it can inhibit phosphatidylcholine synthesis (Barbee and Hartung, 1979Go; Hoffman et al., 1983Go).

The third important feature of the biochemical effects of DEA is that the inhibition of choline uptake and the altered utilization of choline are eliminated in the presence of excess choline, indicating that the actions of DEA are competitive, and hence, reversible in nature. In the presence of excess choline, the simple diffusion of choline into cells likely overcomes the ability of DEA to inhibit choline transport, and in the presence of excess choline, the formation of CDP choline is favored over the formation of CDP derivatives of DEA. The competitive characteristics of these effects also suggest that there is a critical concentration of DEA that must be attained to disrupt choline homeostasis.

It is well recognized that choline deficiency increases spontaneous carcinogenesis in rodents (Ghoshal and Farber, 1984Go; Mikol, 1983; Newberne et al., 1982Go; Newberne and Rogers, 1986Go) and that choline deficiency may also promote liver tumor formation (Lombardi and Shinozuka, 1979Go; Sawada et al., 1990Go; Sells et al., 1979Go; Yokoyama et al., 1985Go). In these studies, choline is typically removed from the diet, creating an intentional nutritional deficit that rapidly reduces hepatic concentrations of PC precursors and subsequently alters cellular 1-carbon metabolism (Christman, 1995Go; Wainfan and Poirer, 1992Go; Zeisel et al., 1989Go; Zeisel and Blusztajn, 1994Go). In the case of DEA, its ability to inhibit choline uptake and compete for the utilization of choline may create an intracellular biochemical condition resembling choline deficiency. If true, animals exposed to DEA would be expected to show hepatic changes consistent with those observed in animals intentionally deprived of choline. To this end, it has recently been reported that when administered for 2 weeks, a carcinogenic dose of DEA (160 mg/kg) significantly reduced hepatic concentrations of choline, phosphocholine, glycerophosphocholine, and phosphatidylcholine in mice (Stott et al. 1999Go). In this report, phosphocholine levels were depleted to less than 20% of control values, and as the intracellular storage form of choline, phosphocholine levels are reduced most dramatically in dietary choline deficiency (Pomfret et al., 1990Go). These data provide the first evidence that, at a dosage known to be carcinogenic, the early biochemical effects of DEA are consistent with those commonly described in choline deficiency, despite the presence of adequate dietary choline.

Collectively, the effects of DEA described here for cultured mammalian cells appear to be directly relevant to in vivo exposure to DEA, and these changes are consistent with the development of choline deficiency. In this regard, the findings that the cellular and biochemical effects of DEA are concentration dependent and prevented by excess choline are particularly important with respect to accurate hazard identification and risk assessment. For example, dermal application of DEA caused liver tumors in mice, but not in rats (NTP, 1999). Disposition data indicate that DEA is less readily absorbed across rat skin than mouse skin and hence, the resulting blood and tissue concentrations of DEA are lower in rats than in mice (Mathews et al., 1997Go). This fact suggests that, in rats, the levels of DEA attained may not reach concentrations necessary to markedly alter choline homeostasis. If true, species differences in tumor susceptibility may simply be a function of the internal dose of DEA, and the lack of carcinogenicity in the rat may be the most appropriate data by which to qualitatively and quantitatively establish the human cancer risk associated with DEA exposure. Alternatively, species differences in tumor susceptibility may also reflect the sensitivity of the B6C3F1 mouse strain to hepatocarcinogenesis (Goodman et al., 1991Go). Clearly, more work to determine the biochemical and molecular changes observed with DEA treatment, the species specificity and dose-response relationships describing these effects, and the accurate determination of human exposure to DEA is warranted.

In vitro cell transformation systems were developed in efforts to simulate the carcinogenic process in cultured cells. An extensive body of literature now demonstrates that cellular changes occurring during morphologic transformation closely parallel multistage chemical carcinogenesis in vivo (Barrett et al., 1984Go; Leboeuf et al., 1999). As a result, these systems are not only useful for studying the molecular and cellular mechanisms of carcinogenesis, but are also gaining in utility as models for predicting the carcinogenic potential of chemicals (IARC, 1999). The present studies combined the predictive utility of the SHE cell transformation assay with its mechanistic attributes to determine whether the carcinogenic effects of DEA are secondary to the development of choline deficiency. The results demonstrate that DEA increases morphologic transformation frequency in SHE cells, and that this transformation occurs along with modulation of the intracellular availability of choline, reduction in the synthesis of choline-containing phospholipids, and the incorporation of DEA into cellular phospholipids. However, choline supplementation blocks the biochemical effects of DEA and prevents cell transformation, suggesting a relationship between the effects of DEA on intracellular choline availability and utilization and its ability to transform cells. Thus, the results of the present studies provide a plausible mechanism to explain the morphologic transformation and support the conclusion that the carcinogenic effects of DEA are likely caused by biochemical perturbations consistent with choline deficiency.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the contribution of Dr. Bob Binder for suggesting choline supplementation in the cell transformation assays and Dr. Steven Robison for his wisdom and helpful suggestions in the overall conduct of this work. The technical assistance of Mr. Dave Gibson in the conduct of the SHE cell transformation assays is also recognized and appreciated.


    NOTES
 
1 To whom correspondence should be addressed at Miami Valley Laboratories, Procter and Gamble Co., P.O. Box 538707, Cincinnati, OH 45253. Fax: (513) 627-1908. E-mail: lehmanmckeemanld{at}pg.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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