* Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 84674; and Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275997641
Received January 9, 2004; accepted February 14, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: triethanolamine; mechanism; choline deficiency.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The secondary amine analogue of TEA, diethanolamine (DEA), is a minor impurity in most TEA formulations. DEA has also been reported to cause liver tumors in mice, albeit a more pronounced response, but not in rats under a similar study design (NTP, 1999b) as used in the TEA studies. Subsequent investigations of the possible mode of tumorigenesis of DEA resulted in the elucidation of a DEA-induced choline-deficiency-based mechanism for tumor formation in treated mice (Lehman-McKeeman et al., 2002
; Lehman-McKeeman and Gamsky, 2000
; Stott et al., 2000a
). Significantly, the mechanism by which choline deficiency can cause tumors in rodents has been well characterized, and a general lack of sensitivity of higher mammals to develop this deficiency has been identified (Lehman-McKeeman et al., 2002
; Stott et al., 2000a
; reviewed by Zeisel and Blusztajn, 1994
). DEA was shown both to upset a primary route of choline synthesis in cells by its displacement of ethanolamine in synthesis of phosphatidylethanolamine and to interfere with the uptake of choline across the cell membrane (Barbee and Hartung, 1979
; Lehman-McKeeman and Gamsky, 1999
). Species-specific increases in cell proliferation were characterized by Kamendulis et al. (2002
, 2003
), and tumorigenic dosages of DEA also depressed the methylation potential of liver in mice (Lehman-McKeeman et al., 2002
; Stott et al., 2000b
).
Treatment with a variety of alkylamines and alkanolamines has been reported to alter phospholipid synthesis and choline incorporation into phospholipids of cultured hepatocytes and/or cell lines (Akesson, 1977; Borman, 1982
; Glaser et al., 1974
). These findings suggest the possibility that TEA, like DEA, may inhibit the uptake of choline by liver in treated mice, resulting in choline deficiency and a similar mode of tumorigenesis as with DEA. Thus, this study examined the potential of TEA to alter choline levels in mice and rats at dose levels and means of administration utilized in the NTP (1999a
; 2003
) bioassays.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and dosing.
Female B6C3F1/CrlBR® (B6C3F1) mice and CDF®(Fischer 344/CrlBR) (CDF) rats were obtained from Charles River Laboratories Inc. (Portage, MI). The laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, and the Laboratory Animal Care and Use Committee approved all procedures used in the study. Animals were housed one per cage in appropriate stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Animals were provided LabDiet® Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) in pelleted form in a hanging feeder and municipal water from a pressure-activated nipple-type watering system ad libitum. This diet contains approximately 1800 ppm choline. Following acclimation, animals (812 weeks of age) were stratified using preexposure body weights and were randomly assigned to treatment groups using a computer program and identified via subcutaneously implanted transponders (BioMedic Data Systmens, Seaford, Delaware). Animals were observed twice daily to evaluate health status and weighed weekly during the study.
An area on the back of each mouse or rat, between the scapulae and stretching posterior approximately halfway to the ileum, was clipped free of hair at least 24 h prior to initiation of dosing. This area was reclipped over the course of the dosing period, as needed, at least 24 hours prior to resumption of dosing. Care was taken to avoid abrasian or nicking of skin. Dosing was as described by NTP (1999a; 2003
), by applying ("painting") solutions or vehicle (acetone) directly on the skin using a blunt syringe. Dose solutions in acetone vehicle were applied at a volume of 4 ml/kg (250 mg/ml) in the first mouse trial, 2 ml/kg (5500 mg/ml) in the second mouse trial, and 0.5 ml/kg (500 mg/ml) in the rat trial. The exposure site was not occluded, nor was any restraining device employed to prevent grooming. An evaluation of potential irritancy of the dosing solutions to the application site skin was made weekly using a standard evaluation scheme.
In vivo study design and choline analysis.
Two in vivo evaluations of the effect of dermally administered TEA upon hepatic choline and metabolites were undertaken utilizing dosages used in the NTP bioassay (NTP, 2003). In the first trial, groups of ten mice were administered 0 (acetone vehicle) or 1000 mg/kg/day TEA 5 days/week for 3 weeks. In the second trial, groups of eight mice were administered 0 (acetone), 10, 100, 300, or 1000 mg/kg/day, and rats were administered 0 (acetone) or 250 mg/kg/day TEA 5 days/week for 3 weeks. Dose levels encompassed those used in the NTP bioassays of TEA in mice (NTP, 2003
) and rats (NTP, 1999a
). Animals were sacrificed on the last day of exposure approximately 24 hours following the last dermal dosing. Mice and rats were rapidly anesthetized with CO2, decapitated, and briefly exsanguinated, and livers were rapidly excised and snap-frozen in liquid nitrogen. Livers were stored at 80°C and shipped on dry ice to Dr. Steven Zeisel's laboratory at the University of North Carolina (Chapel Hill, NC) for analysis.
Hepatic choline and its metabolites, PCho and betaine, were quantitated using the liquid chromatography (LC) electrospray ionization (ESI) isotope dilution mass spectrometry method outlined by Koc et al. (2002). Briefly, weighed samples of liver were extracted with a methanol/chloroform solution, appropriate deuterated internal standards were added, and the aqueous fraction components were separated using LC, followed by ESI and monitoring of selected ions by mass spectrometry. Data collection and analysis was as described using XCalibur® software.
3H-Choline uptake in vitro.
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie (Oak Ridge National Laboratory, Oak Ridge, TN) was used. Cells were grown in Ham's F-12 nutrient mix fortified with 5% (V/V) heat-inactivated, dialyzed fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 0.25 µg/ml), and an additional 2 mM L-glutamine under a 5% CO2 atmosphere at 37°C. CHO cells were seeded at a density of 2 x 105/well in 1 ml of culture medium in 24-well culture plates and cultured overnight. Dose solutions were prepared gravimetrically by mixing high purity TEA or DEA in culture medium, adjusting the pH to 7.4 to achieve concentrations of approximately 0.673.4 mM TEA and 0.051.9 mM DEA. The highest concentration of DEA as an impurity in TEA at the concentrations tested was 0.2 mg/ml. 3H-Choline (5 mCi/well) was added directly to actively growing (nonconfluent) cultures, which were then incubated under a 5% CO2 atmosphere at 37 °C for an additional 10 min. The final concentration of choline in the media of treated cultures was approximately 367 mM. Choline uptake was stopped by the addition of 1 ml of ice-cold PBS/well. Excess 3H-choline was removed by repeated washing of the wells with PBS. Cells were then trypsinized, pelleted by centrifugation at 2000 x g for 5 min, and solubilized in 0.1 N sodium hydroxide. Wells containing media only served as controls. The protein concentration of each solubilized culture was determined using a BCA assay (Pierce, Rockford, IL), and 3H content was quantitated using liquid scintillation counting. Uptake was calculated as total recovered radioactivity per mg protein.
Statistics.
Data were evaluated by Bartlett's test (alpha = 0.01) for equality of variances, followed by a parametric analysis of variance (ANOVA) (Steel and Torrie, 1960). If significant at alpha = 0.05, a Dunnett's test (alpha = 0.05) was conducted (Winer, 1971
). In addition, a linear orthogonal polynomial contrast was used to test for linear trend in dose-response data using a significance value of 0.05 (Trial II mouse data) (Winer, 1971
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Vivo Choline and Choline-Related Metabolites
In the initial mouse trial (Trial I), a 1000 mg/kg/day TEA dosage caused statistically identified decreases in betaine (26%) and PCho (35%) levels relative to vehicle treated controls (Fig. 1). A smaller decrease in hepatic choline concentration (13%) was also observed, which was not statistically identified. In a subsequent dose-response experiment (Trial II mice), all three measured parameters were statistically identified by Trend Test as changing over the dose range, despite a noticeable degree of variability in the data (Table 1). PCho levels were decreased by 1820% at 100300 mg/kg/day and by 42% at 1000 mg/kg/day compared to controls. Hepatic betaine levels were also decreased across most dosages, with minimal levels observed at the high dosage (29% decrease), and choline levels of high-dose-group mice were depressed by 35% compared to controls. Pairwise statistically significant changes were limited to high-dose groups. Administration of 250 mg/kg/day TEA to male CDF rats failed to cause a significant change in any measured parameter.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Administration of TEA to B6C3F1 mice in the present study was clearly shown to decrease hepatic levels of the primary storage form of choline, PCho, and its oxidation product, betaine (Pelech and Vance, 1984). In both mouse Trial I and II, significant decreases in PCho levels (35 and 42%, respectively) and betaine levels (26 and 29%, respectively) occurred at 1000 mg/kg/day relative to controls. Decreases in choline levels also occurred at this high dosage, but to a much greater degree (35% decrease) in the second trial than in Trial I (13% decrease). A dose-related, albeit somewhat variable, decrease in PCho levels was also observed in the second trial, ranging from 18% to 42% over the three dose levels utilized in the NTP bioassay (100, 300, and 1000 mg/kg/day). The variability in measured parameters is not surprising, given the rapid metabolism of choline (Pelech and Vance, 1984
) and the potential variability inherent in dosing via skin painting. However, it is noteworthy that control values of choline, PCho, and betaine were similar to those of more recent reports (Lehman-McKeeman et al., 2002
; Stott et al., 2000b
). Significantly, no consistent evidence of decreases in measured parameters occurred in mice administered 10 mg/kg/day TEA, nor were changes observed in female CDF rats administered a maximum tolerated and yet nontumorigenic dose level of 250 mg/kg/day.
Decreases in hepatic PCho, betaine, and choline levels observed at 1000 mg/kg/day in the second trial were more pronounced than in the first trial. Trial I involved administration of a relatively pure sample of TEA having only approximately 0.04% DEA impurity. The resulting dosage of DEA in Trial I was thus only approximately 0.4 mg/kg/day DEA, well below the no effect level of 10 mg/kg/day DEA for demonstrable changes in choline levels in mice under a similar study design by Lehman-McKeeman et al. (2002). In the subsequent Trial II, a sample of TEA containing approximately 0.45% DEA was used to provide comparative data. A generous gift from NTP, this latter TEA was used in the most recent NTP (2003)
bioassay and resulted in coadministration of a maximal 4.5 mg/kg/day DEA dose. While still below the no effect level for DEA in the Lehman-McKeeman et al. (2002)
study, an additive effect with that of TEA in the present study appears likely.
The loss of betaine in TEA-treated mice in the present study is particularly significant because it represents a loss of a choline metabolite that is central to the synthesis of S-adenosylmethionine (SAM), a principle methylating agent for biosynthetic pathways and maintenance of critical gene methylation patterns. Loss of hepatic betaine and SAM has also been identified in choline-deficient rodents (Zeisel and Blusztajn, 1994). Similar changes in hepatic betaine levels were observed by Lehman-McKeeman et al. (2002)
. Data from this latter study revealed an association between an approximately 20% decrease in hepatic betaine levels and decreases in SAM levels in B6C3F1 but not C57BL/6 mice treated with ethanol and/or a tumorigenic dose level of DEA. The former strain is relatively sensitive, and the latter strain resistant to spontaneous liver tumor formation, suggesting a possible link between these factors in the relatively sensitivity B6C3F1 mouse strain.
While sharing a mode of tumorigenic action, it is obvious that overall TEA is less potent than DEA at causing choline depression in mice. Lehman-McKeeman et al. (2002) reported hepatic PCho levels depressed by 2150% in B6C3F1 mice administered tumorigenic dose levels of 40160 mg/kg/day DEA for 4 weeks. A depression of approximately 80% in hepatic PCho levels was obtained at 160 mg/kg/day DEA by Stott et al. (2000a
,b
) under similar conditions. Not surprisingly, the choline depressive responses obtained with these two compounds reflect their relative tumorigenic potencies. TEA resulted primarily in an elevation in single occurrences of benign liver tumors in one sex of B6C3F1 mice, while DEA caused multiple occurrences of both benign and malignant tumors in both sexes of mice (NTP 1999b
; 2003
).
DEA is known to affect choline levels by inhibiting the uptake of choline by cells (Lehman-McKeeman and Gamsky, 1999) and by competition with ethanolamine in phosphatidylethanolamine synthesis (Barbee and Hartung, 1979
; Lehman-McKeeman and Gamsky, 1999
; Mathews et al., 1995
), effectively limiting a primary biosynthetic source of choline in mature animals. Effects of TEA upon the uptake of 3H-choline by CHO-K1 cells were observed in the present study. Consistent with the relative potency in depleting cellular choline pools in vivo, TEA was much less potent than DEA at inhibiting 3H-choline uptake, with maximal effects of approximately 6070% of control obtained at 1.342.68 mM TEA versus approximately 74% of control at 0.19 mM DEA. Effects of TEA were not attributed to the small amounts of DEA impurity in the test material. The concentrations of DEA needed to cause a minimal effect upon 3H-choline uptake in DEA assays was 36- to180-fold higher than the levels in TEA-treated cultures. In addition, TEA is unlikely to alter de novo synthesis of choline for several reasons. No conversion to DEA was observed in mice dosed with 14C-TEA, and unlike DEA, no accumulation of TEA in liver tissues of mice occurred, as would be expected if metabolic incorporation at the expense of ethanolamine into phosphatidylethanolamine was occurring (Mathews et al., 1997
; D. Rick, unpublished data; Stott et al., 2000c
). Lehman-McKeeman (personal communication) has also demonstrated a lack of effect of TEA treatment upon synthesis rates of phosphatidylethanolamine in cultured CHO-K1 cells. Finally, the much broader spectrum of organ toxicity observed in test animals with DEA than with TEA suggests a fundamental difference in the metabolism of the two materials (reviewed by Knaak et al., 1997
; NTP, 2003
).
It was concluded that TEA induces changes in liver of mice consistent with a choline-deficiency mode of tumorigenesis, as elucidated by S. Zeisel and coworkers (reviewed by Zeisel and Blusztajn, 1994) and most recently demonstrated for DEA in mice by Lehman-McKeeman et al. (2002)
and Stott et al. (2000a)
. This effect appears to be a property of TEA exclusive of any DEA impurity; however the latter may also contribute to choline depletion in treated mice. Tumor formation requires the chronic depression of choline pools resulting in a relatively well-characterized sequence of biochemical, cytological, and genomic changes. Significantly, this nongenotoxic mode of tumorigenesis displays thresholds and differences in interspecies sensitivity, with higher primates being much more resistant than rodent species.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed. E-mail: wstott{at}dow.com
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbee, S. J., and Hartung, R. (1979) The effect of diethanolamine on hepatic and renal phospholipid metabolism in the rat. Toxicol. Appl. Pharmacol. 47, 421430.[ISI][Medline]
Borman, L. S. (1982). Modulation of mammalian cell growth by a choline analog, N-isopropylethanoamine. In Vitro 18, 129140.[ISI][Medline]
Glaser, M., Ferguson, K. A., and Vagelos, P. R. (1974). Manipulation of the phospholipid composition of tissue culture cells. Proc. Nat. Acad. Sci. U.S.A. 71, 40724076.[Abstract]
Kamendulis, L. M., Smith, D. J., Jiao, Z., and Klaunig, J. E. (2003). Species differences in the induction of hepatocellular DNA synthesis by diethanolamine. The Toxicol. 72, Abstr. No. 1155.
Kamendulis, L. M., Smith, D. J., and Klaunig, J. E. (2002). Role of choline depletion in diethanolamine induced DNA synthesis in mouse hepatocytes. The Toxicol. 66, Abstr. No. 1507.
Knaak, J. B., Leung, H.-W., Stott, W. T., Busch J., and Bilsky, J. (1997). Toxicology of mono-, di-, and triethanolamine. Rev. Environ. Contam. Toxicol. 149, 186.[Medline]
Konishi, Y., Denda, A., Uchida, K., Emi, Y., Ura, H., Yokose, Y., Shiraiwa, K., and Tsutsumi, M. (1992). Chronic toxicity carcinogenicity studies of triethanolamine in B6C3F1 mice. Fundam. Appl. Toxicol. 18, 2529.[ISI][Medline]
Koc, H., Mar, M.-H., Ranasinghe, A., Swenberg, J. A., and Zeisel, S. H. (2002). Quantitation of choline and its metabolites in tissues and foods by liquid chromatography/electrospray ionization-isotope dilution mass spectrometry. Anal. Chem. 74, 47344740.[CrossRef][ISI][Medline]
Lehman-McKeeman, L. D., and Gamsky, E. A. (1999). Diethanolamine inhibits choline uptake and phosphatidylcholine synthesis in Chinese Hamster ovary cells. Biochem. Biophys. Res. Comm. 262, 600604.[CrossRef][ISI][Medline]
Lehman-McKeeman, L. D., and Gamsky, E. A. (2000). Choline supplementation inhibits diethanolamine-induced morphological transformation in Syrian Hamster embryo cells: Evidence for a carcinogenic mechanism. Toxicol. Sci. 55, 303310.
Lehman-McKeeman, L. D., Gamsky, E. A., Hicks, S. M., Vassallo, J. D., Mar, M.-H., and Zeisel, S. H. (2002). Diethanolamine induces hepatic choline deficiency in mice. Toxicol. Sci. 67, 3845.
Mathews, J. M., Garner C. E., Black, S. L., and Matthews H. B. (1997). Diethanolamine absorption, metabolism and disposition in rat and mouse following oral, intravenous and dermal administration. Xenobiotica 27, 733746.[CrossRef][ISI][Medline]
Mathews, J. M., Garner, C. E., and Matthews H. B. (1995). Metabolism, bioaccumulation, and incorporation of diethanolamine into phospholipids. Chem. Res. Toxicol. 8, 625633.[ISI][Medline]
National Toxicology Program (NTP) (1999a). Toxicology and Carcinogenesis Studies of Triethanolamine (CAS No. 102716) in F344/N Rats and B6C3F1 Mice (Dermal Studies), NTP TR 449, NIH Publication No. 003365.
National Toxicology Program (NTP) (1999b). Toxicology and Carcinogenesis Studies of Diethanolamine (CAS No. 111422) in F344/N Rats and B6C3F1 Mice (Dermal Studies), NTP TR 478, NIH Publication No. 973968.
National Toxicology Program (NTP) (2003). Toxicology and Carcinogenesis Studies of Triethanolamine (CAS No. 102716) in F344/N Rats and B6C3F1 Mice (Dermal Studies), NTP TR 518, NIH Publication No. 034452.
Pelech, S. L., and Vance, D. E. (1984). Regulation of phosphatidylcholine biosynthesis. Biochim. Biophys. Acta 779, 217251.[ISI][Medline]
Steel, R. G. D., and J. H. Torrie. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York.
Stott, W. T., Bartels, M. J., Brzak, K. A., Mar, M.-H., Markham, D. A., Thornton, C. M., and Zeisel, S. H. (2000a). Potential mechanisms of tumorigenic action of diethanolamine in mice. Toxicol. Lett. 114, 6775.[CrossRef][ISI][Medline]
Stott, W. T., Bartels, M. J., Brzak, K. A., Mar, M.-H., Markham, D. A., Thornton, C. M., Kan, L., Curry, S., Purdon, M., and Zeisel, S. H. (2000b). Potential mechanisms of tumorigenic action of diethanolamine in mice. The Toxicol. 54, Abstr. No. 1022.
Stott, W. T., Waechter, J. M., Jr., Rick, D. L., and Mendrala, A. L. (2000c). Absorption, distribution, metabolism and excretion of intravenously and dermally administered triethanolamine in mice. Food Chem. Toxicol. 38, 10431051.[CrossRef][ISI][Medline]
Winer, B. J. (1971). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York.
Zeisel, S. H., and Blusztajn, J. K. (1994). Choline and human nutrition. Ann. Rev. Nutr. 14, 269296.[CrossRef][ISI][Medline]
|