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Potential mechanisms of tumorigenic action of diethanolamine in mice
Toxicology Letters 114 (2000) 67 – 75 www.elsevier.com/locate/toxlet
Potential mechanisms of tumorigenic action of diethanolamine in mice W.T. Stott a,*, M.J. Bartels a, K.A. Brzak a, M.-H. Mar b, D.A. Markham a, C.M. Thornton a, S.H. Zeisel b a
Toxicology and En6ironmental Research and Consulting, Dow Chemical Company, Bldg. 1803, Midland, MI 48674, USA b Department of Nutrition, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC, USA Received 7 September 1999; received in revised form 21 October 1999; accepted 21 October 1999
Abstract Diethanolamine (DEA), a secondary amine found in a number of consumer products, reportedly induces liver tumors in mice. In an attempt to define the tumorigenic mechanism of DEA, N-nitrosodiethanolamine (NDELA) formation in vivo and development of choline deficiency were examined in mice. DEA was administered with or without supplemental sodium nitrite to B6C3F1 mice via dermal application (with or without access to the application site) or via oral gavage for 2 weeks. Blood levels of DEA reflected the dosing method used; oral greater than dermal with access greater than dermal without access. No NDELA was observed in the urine, blood or gastric contents of any group of treated mice. Choline, phosphocholine and glycerophosphocholine were decreased 562–84% in an inverse relation to blood DEA levels. These data demonstrated a lack of NDELA formation in vivo at tumorigenic dosages of DEA but revealed a pronounced depletion of choline-containing compounds in mice. It is suggested that the latter effect may underlie DEA tumorigenesis in the mouse. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mechanism tumorigenesis; Diethanolamine; Choline deficiency; N-Nitrosodiethanolamine
1. Introduction Diethanolamine (DEA) has found extensive use in the production of amides of carboxylic acids for use in a variety of products. The oncogenic potential of DEA has been examined in a skin painting bioassay conducted by the National Toxicology Program (NTP, 1997). The method of * Corresponding author. E-mail address: [email protected] (W.T. Stott)
administration allowed dosed animals free access to the application site during grooming activities, resulting in the ingestion of an indeterminate amount of the applied dose. An increased incidence of liver tumors (adenomas, carcinomas and hepatoblastomas) were observed in mice administered ] 40 mg/kg per day DEA and of kidney adenomas in male mice administered 160 mg/kg per day. In contrast, no tumorigenic response was noted in male or female rats administered 5 64 mg/kg per day DEA and 5 32 mg/kg per day,
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respectively, nor did DEA promote tumors in a Tg.AC transgenic mouse assay (Tennant et al., 1996). DEA has been shown to be negative in a number of genotoxicity assays (reviewed by Knaak et al., 1997). A reported positive response in a SHE cell transformation assay (Kerckaert et al., 1996) was eliminated upon supplementation of culture media with choline (Lehman-McKeeman, personal communication). These data suggest that DEA caused tumors in B6C3F1 mice via a secondary, possibly nongenotoxic, mechanism. As a secondary amine, DEA may undergo nitrosation to form the nitrosamine N-diethanolamine (NDELA) (reviewed in ECETOC, 1990). NDELA is mutagenic in vitro and reportedly causes liver tumors in rats at ] 2 mg/kg per day (ECETOC, 1990). Formation of NDELA in vivo has been measured directly or inferred in rats administered relatively high, often toxic, dosages of both DEA and nitrite (Preussmann et al., 1981; Konishi et al., 1986; Yamamoto et al., 1995). It has also been suggested that the toxicity of DEA may be related to its metabolic incorporation into phospholipids which are essential to membrane structure and function (Barbee and Hartung, 1979; Mathews et al., 1995). Barbee and Hartung (1979) reported a significant inhibition of hepatic phosphatidylcholine (PtdCho) synthesis in rats administered 330 mg/kg per day DEA via drinking water for 1 week. Mathews et al. (1995) further identified the incorporation of DEA, and possibly its methylated metabolites, into the hepatic phosphatidylethanolamine (PtdEtn) biosynthesis pathway. Mathews et al. (1995) also observed a significant portion of incorporated DEA in ceramide phospholipid headgroups. Alterations in phospholipid metabolism may disrupt choline metabolism which, in-turn, may have multiple effects including altered membrane function, rates of cell proliferation and apoptosis, increased protein kinase C activity, and decreased DNA methylation (reviewed by Zeisel and Blusztajan, 1994). Choline deficiency is a known tumor promotor, especially of nitrosamine-initiated carcinogenesis in rodents (DeCarmargo et al., 1985; Newberne and Rogers, 1986) and choline deficiency by itself is the only known nutritional deficiency considered carcinogenic in rodents (Newberne and Rogers, 1986).
This study was undertaken to evaluate DEA-related NDELA formation and related changes in choline-containing compounds in mice.
2. Methods
2.1. Materials Materials were obtained as follows: DEA of 99.7% purity from Dow Chemical Company (Plaquemine, LA); NDELA (98% purity) from Chem Service (West Chester, PA); Deuterated (D8)-NDELA from ISOTEC (Miamisburg, OH); 14 C-labeled internal standards for choline, (phosphocholine (PCho) and glycerophosphocholine (GPCho) from New England Nuclear (Boston, MA) or ICN Biomedicals (Irvine, CA); PtdCho, PtdEtn and sphingomyelin (SM) standards from Sigma (St. Louis, MO); and Silica-gel coated thinlayer-chromatography plates from J.T. Baker Chemical Company (Phillipsburg, NJ). D8-DEA was prepared by heating a solution of 0.587 g (4.55 mmol) 1,1,2,2-D4-bromoethanol (Cambridge Isotopes, Andover, MA) and concentrated ammonium hydroxide (0.3 ml, 4.5 mmol) in 1.5 ml 2-propanol at 60°C for 1.7 h. Upon cooling, the mixture was adjusted to pH 12–13 (5 N NaOH) and extracted with methylene chloride (1× 2 ml) and taken to dryness (nitrogen stream) to afford 0.48 g of the crude product. GC/MS analysis found 13% as the desired D8-DEA, with the remainder as D4-monoethanolamine, D12-triethanolamine and solvent.
2.2. Animals Male B6C3F1 mice (approximately 28–30 g) were obtained from Charles River (Portage, MI). Mice were singly housed in stainless steel cages containing a feeder and water bottle equipped with a sipper tube under environmentally-controlled conditions.1 Mice were assigned to control and treatment groups using a computer-driven randomization program and were identified using 1 Laboratory accredited by Association for Assessment and Accreditation of Laboratory Animal Care International.
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ear tags. Animals were provided Purina Certified Rodent Lab Diet c5002 (Purina Mills, St. Louis, MO) in pelleted form ad libitum during the prestudy and dosing periods. Feed and water nitrate levels were approximately 22 and 1.6 mg/g, respectively. Nitrite levels were B2 mg/g. Animals were weighed weekly.
2.3. Design Two trials were conducted, each having four dose groups of five to six B6C3F1 mice; (1) controls which were clipped and depilated only; (2) 160 mg/kg per day DEA via dermal application with no access to the application site; (3) 160 mg/kg per day DEA via dermal application with access to the application site; and (4) 160 mg/kg per day DEA via oral gavage. Animals were dosed 7 days/week for 2 weeks. In the first trial, no supplemental nitrite was co-administered mice to mimic standard laboratory conditions. Sample collection was limited to urine, which was collected after approximately 1 and 2 weeks of dosing, and blood, which was collected at sacrifice. In the second trial, supplemental sodium nitrite was provided to all groups via their drinking water (140 ppm; approximately 40 mg/kg per day) to further evaluate NDELA formation in vivo under non-bolus dosing situations. Sample collection in the second trial was limited to blood, gastric contents and liver. Preliminary experiments to establish peak blood NDELA concentrations and dose-response of NDELA formation in blood and gastric contents of mice (method validation) were undertaken using oral gavage dosing of DEA plus sodium nitrite. Urine, blood and gastric contents were analyzed for NDELA and blood was also analyzed for DEA. Livers were excised in the second trial and the hepatic concentrations of a number of phospholipids, choline and cholinemetabolites were determined.
2.4. Dosing In both trials, animals were dosed at a constant level of 160 mg/kg per day based upon the most recent body weights. Oral gavage dosages were prepared in distilled water at a dosing volume of
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10 ml/kg and were delivered using a 1 cc tuberculin syringe equipped with a disposable gavage needle. Dermal application sites consisted of a roughly 4 cm2 area running from the interscapular region several cm posterior of this. The area was clipped free of fur, depilated using a commercial depilatory, rinsed with a wet paper towel and blotted dry prior to initiation of dosing and subsequently on an as-needed basis. Care was taken during the preparation of the site to avoid irritation of the skin. Dermal administration mimicked the NTP (1997) bioassay of DEA in that a 90 mg/ml concentration in ethanol was administered at a volume of 1.78 ml/kg. Mice were dosed by spreading a known volume of test material about the prepared target site using a micropipetter equipped with a blunted tip. In the first trial, access to the application site was prevented by use of an ADVANCED CURAD® AQUA-PROTECT™ bandage (38 mm× 38 mm) placed over the dosing site. In the second trial, a Teflon® collar consisting of a 20 mm band of flexible Teflon, as described by Wahle et al. (1998) was fitted about the necks of mice to prevent animals from grooming. Another group was allowed access to the application site during normal grooming activities. A group of six untreated, but clipped and depilated, mice served as controls in each trial. In the second trial, sodium nitrite spiked drinking water was changed every 3 days during the dosing period.
2.5. Sample collection In the first trial, urine was collected on test days 5–7 and 12–14 by placing mice in a Roth-type glass metabolism cage. Urine was collected on dry ice and was acidified to prevent further NDELA production. In both trials, mice were sacrificed approximately 1–2 h after the final dose. Mice were anesthetized with methoxyflurane and exsanguinated by cardiac puncture. In the second trial, livers and stomachs of all mice were then excised, stomachs were split open and gastric contents recovered. Gastric contents, liver and blood samples were then weighed, frozen in liquid nitrogen and stored at − 80°C. Blood was analyzed for DEA. Urine, blood and gastric contents were
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analyzed for NDELA concentration and liver was analyzed for PtdCho, PtdEtn, choline, SM, PCho, and GPCho.
2.6. DEA and NDELA analyses DEA was determined in blood and NDELA was determined in urine, gastric contents and blood of all mice using gas chromatographymass spectrometry (GC-MS). Samples for DEA or NDELA analysis were weighed and spiked with a known amount of a D8-DEA and D8NDELA internal standard, respectively, to ensure accurate quantitation. DEA whole blood samples were then pH-adjusted with 2.5 N KOH and derivatized with pentafluorobenzoyl chloride, extracted into toluene and analyzed. NDELA urine samples were acidified with 1 N HCl while gastric contents and blood samples were fortified with 20% NaCl and ammonium carbonate ( 25 mg). All NDELA samples were subsequently extracted into ethyl acetate, concentrated to dryness with N2, and derivitized with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) in toluene. Derivatized DEA and NDELA were then analyzed using GC-MS to ensure structural confirmation of DEA and NDELA present in the appropriate samples. Separations were achieved on a DB5ms capillary column (J&W Scientific, Folsom, CA) and the mass spectrometer was operated in the negative ion/chemical ionization mode (CH4) monitoring ions 332 (NDELA) and 340 (D8NDELA), or 687 (DEA) and 695 (D8-DEA). Analytical standards were prepared in control matrix. Limits of detection (LOD) of NDELA were determined to be 324 ng/g in urine, 1.8 ng/g in gastric contents, and 5 and 1.5 ng/g in blood in the first and second trials, respectively.
Dyer, 1959), spiked with appropriate [14C]methyl and [2H]methyl labeled internal standards to permit peak selection and calculation of recovery efficiency, and separated into organic and aqueous phases. Hydrophilic choline metabolites were then separated by high performance liquid chromatography and quantitated using gas chromatography-mass spectroscopy. PtdCho, PtdEtn and SM were analyzed using the methods outlined by Svanborg and Svennerholm (1961) and Yen et al. (1999). Briefly, following extraction, hydrophobic metabolites were separated using thin layer chromatography (TLC), recovered by scraping appropriate bands and quantitated along with authentic phospholipid standards using a phosphorous assay.
2.8. Clinical chemistry (second trial) Serum was harvested from collected blood samples and the activies of alanine and aspartate aminotransferases (ALT and AST, respectively) were determined using a Hitachi Model 914 Automatic Analyzer (Boehringer Mannheim, Indianapolis, IN).
2.9. Statistics All measurable parameters were first evaluated for homogeneity of variance (a= 0.01) followed by a one-way analysis of variance (ANOVA) with dose as the factor (Winer, 1971). If the dose effect was significant, a Tukey’s HSD test was conducted at an a= 0.05 (Kramer, 1956).
3. Results
3.1. NDELA analyses 2.7. Choline and phospholipid analyses (second trial) Choline and its metabolites PCho and GPCho were analyzed using the methods outlined by Zeisel et al. (1995) and Pomfret et al. (1989). Briefly, liver samples were extracted (Bligh and
No NDELA was identified in the urine or blood of mice from any treatment group in the absence of supplemental nitrite intake (first trial) or in blood or gastric contents when supplemental nitrite was administered (second trial).
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3.2. Blood DEA analyses The levels of DEA attained in the blood of mice at 1 –1.5 h post-dosing on the final day of the dosing period are shown in Fig. 1. Blood levels ranged from 5 to 7.7 mg/g in a route/method dependent manner. Dermal absorption of DEA administered in an ethanol vehicle over the 2week dosing period resulted in blood levels approximately 65% that obtained from administration of DEA via oral gavage. A trace amount of DEA was also found in the blood of control mice.
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2a–c, choline, PCho and GPCho decreased significantly relative to controls by as much as 64, 84 and 70%, respectively. The smallest decreases occurred in dermal (collared mice) treated mice and the largest in mice administered DEA via oral gavage. PtdCho and PtdEtn levels from the same DEA-treated groups of animals were generally decreased about 20% with the exception of a minimal decrease in PtdCho levels of mice administered DEA via dermal administration (Fig. 2d– e). In contrast, SM levels were increased in treated mice relative to controls (Fig. 2f).
3.3. Choline/phospholipid analyses
3.4. Clinical appearance and serum enzyme le6els
Results of analyses for hepatic PCho, choline, GPCho, PtdCho, PtdEtn, and SM are shown in Fig. 2a–f. A pronounced decrease in choline and its metabolites was observed in the livers of all DEA-treated groups of mice. As shown in Fig.
No clinical signs of toxicity were observed in mice treated with DEA and sodium nitrite (second trial). In addition, ALT and AST levels in DEA treated mice were similar to those of controls (data not shown).
Fig. 1. Blood levels of diethanolamine (DEA) 1–2 h post-dosing in B6C3F1 mice administered 0 (controls) or 160 mg/kg per day DEA via dermal application without access to the dosing site (dermal), dermal application with access during grooming (dermal and oral) or oral gavage (oral). All animals also received approximately 40 mg/kg per day sodium nitrite via their drinking water. [Means and standard deviations (S.D.) of 5 mice/group; * denotes significantly different from controls and different letters denote statistical differences between administration methods; Tukey’s HSD test at an a =0.05.]
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Fig. 2. Hepatic levels of phospholipid and choline-related compounds in B6C3F1 mice administered 0 (controls) or 160 mg/kg per day diethanolamine (DEA) via dermal application without access to the dosing site (Dermal), dermal application with access during grooming (dermal and oral) or oral gavage (oral). All animals also received approximately 40 mg/kg per day sodium nitrite via their drinking water. [Means and standard deviations (S.D.) of 5 mice/group; * denotes significantly different from controls and different letters denote statistical differences between administration methods; Tukey’s HSD test at an a =0.05.]
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Fig. 2. (Continued)
4. Discussion The findings of this study revealed several significant factors impacting on the interpretation of the dermal bioassay of DEA in rodents. These included: Evidence of dermal absorption of DEA in repeatedly dosed mice and the impact of grooming upon absorption of an administered dermal dose of DEA. Lack of significant levels of nitrosamine (NDELA) formation in vivo in mice dosed with tumorigenic dosages of DEA (even orally). Depletion of choline-containing compounds in liver of DEA-treated mice. The comparison of blood levels of DEA obtained in mice administered DEA via strictly dermal means (collared mice) versus oral gavage (Fig. 1) indicates the absorption of dermally applied DEA in mice. Despite the fact that blood data were obtained at a single time point and potential differences between the pharmacokinetics of dermally applied versus oral bolus dosed DEA, blood levels from the former were roughly
65% that obtained from the latter means of administration. This is consistent with the findings of Mathews et al. (1997), who reported that up to 60% of an administered dose of DEA in an ethanol vehicle was absorbed by B6C3F1 mice. The impact of allowing the animals access to the site of application was also apparent as a 35% increase in blood levels obtained in mice allowed access (dermal+ oral) versus those prevented access (wearing collars). Initial analyses of NDELA spiked-urine, blood and gastric ingesta revealed that the latter two matrices provided a more sensitive indicator of nitrosamine formation in vivo than measurements in mouse urine. LODs as low as 1.5–1.8 ng/g in blood and ingesta were attained. Preliminary experiments also established optimal time post-dosing when peak blood levels and measurable NDELA levels in blood and gastric contents would have been expected to occur. No NDELA was found in the urine or blood of male B6C3F1 mice repeatedly administered a carcinogenic dosage of 160 mg/kg per day DEA via dermal application (grooming of application site prevented), a combined dermal and oral (groom-
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ing allowed), or oral gavage in the absence of supplemental nitrite in the first trial. Nitrate consumption via feed and drinking water, estimated to be approximately 5 mg/kg per day, and its presumed conversion to nitrite by gut flora did not support sufficient NDELA synthesis to be detected at the LODs of 324 and 5 ng/g for urine and blood, respectively (first trial). Even upon the addition of supplemental nitrite (170 mg/ml NaNO2) to drinking water, no NDELA was detected in blood or gastric contents of mice repeatedly administered 160 mg/kg per day DEA via oral gavage or skin painting at an LOD of 1.5 – 1.8 ng/g (second trial). Thus, the non-bolus administration of sodium nitrite to mice via drinking water resulted in \2000-fold lower NDELA blood levels than when gavage dosing was employed in preliminary experiments (4.7 mg/ml). Further evidence for the impact of bolus dosing upon NDELA formation comes from Konishi et al. (1986) who recovered 823 mg NDELA in a gastric rinse of rats administered 58 mg/kg DEA and 153 mg/kg sodium nitrite via oral gavage. In contrast, the theoretical maximum total NDELA possible in the ingesta of treated mice in the present study was only 0.35 mg. These data suggest that generation of even low levels of NDELA require relatively large, bolus, dosages of DEA and nitrosating compound(s). As noted, alterations in choline metabolism may have far reaching effects upon the homeostasis of a number of cellular functions including; membrane function, rates of cell proliferation and apoptosis, activity of protein kinase C, and maintenance methylation of DNA (reviewed by Zeisel and Blusztajan, 1994). PCho represents the most labile choline storage pool in rodents while GPCho likely represents a metabolite formed during PtdCho-dependent choline generation (Zeisel et al., 1995). The smaller decreases in PtdCho levels relative to choline and PCho, decrease in the PtdCho metabolite GPCho, and DEA-induced decreases in the rate of PtdCho synthesis reported by Barbee and Hartung (1979) in liver, albeit of rats, suggest that membrane PtdCho is preserved, despite significant depletion of choline precursors by slowing its hydrolysis to GPCho. Thus, a higher demand may be placed upon dietary sources of choline.
The small decreases in hepatic PtdEtn observed in DEA-treated mice (Fig. 2e) likely reflected a portion of DEA incorporated during PtdEtn synthesis (Barbee and Hartung, 1979; Mathews et al., 1995). Again, Barbee and Hartung (1979) had also observed significant decreases in PtdEtn synthesis rates in DEA treated rats. Less clear was the elevation in SM levels directly correlated to blood DEA levels (Fig. 2f). However, this is consistent with the finding by Mathews et al. (1995) that DEA-altered ceramides, and in particular SM, represent the metabolic pool containing a majority of incorporated DEA. Methods utilized to separate and quantitate SM, TLC followed by total phosphorous determination, would not have differentiated between native SM and DEAmodified SM. Ironically, it was reported that choline deficiency was not a factor in the mouse bioassay as fatty liver, a standard histopathological response to dietary choline-deficiency, was not observed in the study (NTP, 1997). The present results indicate that DEA causes this deficiency and demonstrate that biochemical changes can occur without producing fatty liver. Subsequent changes associated with choline deficiency such as hypomethylation of DNA, increased oxidative stress and enhanced S-phase DNA synthesis, which are inturn associated with tumor formation in rodents (reviewed by Goodman et al., 1991), could have accounted for the observed bioassay results. Possible consequences of DEA-induced choline deficiency in mice related to tumorigenesis are presently under study.
Acknowledgements The authors wish to acknowledge the vision and support of Drs H.C. Pitot (Univ. Wisconsin) and L.D. Lehman-McKeeman (Procter & Gamble), Ms L. McFadden (statistics), Mr R. Papciak (Huntsman Chemical Co.), Dr H-W. Leung (Union Carbide) and Dr M. Kayser (formerly of BASF Corp.), and the financial support of the Chemical Manufacturers Association (Alkanolamines Panel) and Cosmetic, Toiletry & Fragrance Association in the design and conduct of
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this work. Dr Zeisel is supported by a grant from NIH (AG 09525).
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Potential mechanisms of tumorigenic action of diethanolamine in mice W.T. Stott a,*, M.J. Bartels a, K.A. Brzak a, M.-H. Mar b, D.A. Markham a, C.M. Thornton a, S.H. Zeisel b a
Toxicology and En6ironmental Research and Consulting, Dow Chemical Company, Bldg. 1803, Midland, MI 48674, USA b Department of Nutrition, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC, USA Received 7 September 1999; received in revised form 21 October 1999; accepted 21 October 1999
Abstract Diethanolamine (DEA), a secondary amine found in a number of consumer products, reportedly induces liver tumors in mice. In an attempt to define the tumorigenic mechanism of DEA, N-nitrosodiethanolamine (NDELA) formation in vivo and development of choline deficiency were examined in mice. DEA was administered with or without supplemental sodium nitrite to B6C3F1 mice via dermal application (with or without access to the application site) or via oral gavage for 2 weeks. Blood levels of DEA reflected the dosing method used; oral greater than dermal with access greater than dermal without access. No NDELA was observed in the urine, blood or gastric contents of any group of treated mice. Choline, phosphocholine and glycerophosphocholine were decreased 562–84% in an inverse relation to blood DEA levels. These data demonstrated a lack of NDELA formation in vivo at tumorigenic dosages of DEA but revealed a pronounced depletion of choline-containing compounds in mice. It is suggested that the latter effect may underlie DEA tumorigenesis in the mouse. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mechanism tumorigenesis; Diethanolamine; Choline deficiency; N-Nitrosodiethanolamine
1. Introduction Diethanolamine (DEA) has found extensive use in the production of amides of carboxylic acids for use in a variety of products. The oncogenic potential of DEA has been examined in a skin painting bioassay conducted by the National Toxicology Program (NTP, 1997). The method of * Corresponding author. E-mail address: [email protected] (W.T. Stott)
administration allowed dosed animals free access to the application site during grooming activities, resulting in the ingestion of an indeterminate amount of the applied dose. An increased incidence of liver tumors (adenomas, carcinomas and hepatoblastomas) were observed in mice administered ] 40 mg/kg per day DEA and of kidney adenomas in male mice administered 160 mg/kg per day. In contrast, no tumorigenic response was noted in male or female rats administered 5 64 mg/kg per day DEA and 5 32 mg/kg per day,
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respectively, nor did DEA promote tumors in a Tg.AC transgenic mouse assay (Tennant et al., 1996). DEA has been shown to be negative in a number of genotoxicity assays (reviewed by Knaak et al., 1997). A reported positive response in a SHE cell transformation assay (Kerckaert et al., 1996) was eliminated upon supplementation of culture media with choline (Lehman-McKeeman, personal communication). These data suggest that DEA caused tumors in B6C3F1 mice via a secondary, possibly nongenotoxic, mechanism. As a secondary amine, DEA may undergo nitrosation to form the nitrosamine N-diethanolamine (NDELA) (reviewed in ECETOC, 1990). NDELA is mutagenic in vitro and reportedly causes liver tumors in rats at ] 2 mg/kg per day (ECETOC, 1990). Formation of NDELA in vivo has been measured directly or inferred in rats administered relatively high, often toxic, dosages of both DEA and nitrite (Preussmann et al., 1981; Konishi et al., 1986; Yamamoto et al., 1995). It has also been suggested that the toxicity of DEA may be related to its metabolic incorporation into phospholipids which are essential to membrane structure and function (Barbee and Hartung, 1979; Mathews et al., 1995). Barbee and Hartung (1979) reported a significant inhibition of hepatic phosphatidylcholine (PtdCho) synthesis in rats administered 330 mg/kg per day DEA via drinking water for 1 week. Mathews et al. (1995) further identified the incorporation of DEA, and possibly its methylated metabolites, into the hepatic phosphatidylethanolamine (PtdEtn) biosynthesis pathway. Mathews et al. (1995) also observed a significant portion of incorporated DEA in ceramide phospholipid headgroups. Alterations in phospholipid metabolism may disrupt choline metabolism which, in-turn, may have multiple effects including altered membrane function, rates of cell proliferation and apoptosis, increased protein kinase C activity, and decreased DNA methylation (reviewed by Zeisel and Blusztajan, 1994). Choline deficiency is a known tumor promotor, especially of nitrosamine-initiated carcinogenesis in rodents (DeCarmargo et al., 1985; Newberne and Rogers, 1986) and choline deficiency by itself is the only known nutritional deficiency considered carcinogenic in rodents (Newberne and Rogers, 1986).
This study was undertaken to evaluate DEA-related NDELA formation and related changes in choline-containing compounds in mice.
2. Methods
2.1. Materials Materials were obtained as follows: DEA of 99.7% purity from Dow Chemical Company (Plaquemine, LA); NDELA (98% purity) from Chem Service (West Chester, PA); Deuterated (D8)-NDELA from ISOTEC (Miamisburg, OH); 14 C-labeled internal standards for choline, (phosphocholine (PCho) and glycerophosphocholine (GPCho) from New England Nuclear (Boston, MA) or ICN Biomedicals (Irvine, CA); PtdCho, PtdEtn and sphingomyelin (SM) standards from Sigma (St. Louis, MO); and Silica-gel coated thinlayer-chromatography plates from J.T. Baker Chemical Company (Phillipsburg, NJ). D8-DEA was prepared by heating a solution of 0.587 g (4.55 mmol) 1,1,2,2-D4-bromoethanol (Cambridge Isotopes, Andover, MA) and concentrated ammonium hydroxide (0.3 ml, 4.5 mmol) in 1.5 ml 2-propanol at 60°C for 1.7 h. Upon cooling, the mixture was adjusted to pH 12–13 (5 N NaOH) and extracted with methylene chloride (1× 2 ml) and taken to dryness (nitrogen stream) to afford 0.48 g of the crude product. GC/MS analysis found 13% as the desired D8-DEA, with the remainder as D4-monoethanolamine, D12-triethanolamine and solvent.
2.2. Animals Male B6C3F1 mice (approximately 28–30 g) were obtained from Charles River (Portage, MI). Mice were singly housed in stainless steel cages containing a feeder and water bottle equipped with a sipper tube under environmentally-controlled conditions.1 Mice were assigned to control and treatment groups using a computer-driven randomization program and were identified using 1 Laboratory accredited by Association for Assessment and Accreditation of Laboratory Animal Care International.
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ear tags. Animals were provided Purina Certified Rodent Lab Diet c5002 (Purina Mills, St. Louis, MO) in pelleted form ad libitum during the prestudy and dosing periods. Feed and water nitrate levels were approximately 22 and 1.6 mg/g, respectively. Nitrite levels were B2 mg/g. Animals were weighed weekly.
2.3. Design Two trials were conducted, each having four dose groups of five to six B6C3F1 mice; (1) controls which were clipped and depilated only; (2) 160 mg/kg per day DEA via dermal application with no access to the application site; (3) 160 mg/kg per day DEA via dermal application with access to the application site; and (4) 160 mg/kg per day DEA via oral gavage. Animals were dosed 7 days/week for 2 weeks. In the first trial, no supplemental nitrite was co-administered mice to mimic standard laboratory conditions. Sample collection was limited to urine, which was collected after approximately 1 and 2 weeks of dosing, and blood, which was collected at sacrifice. In the second trial, supplemental sodium nitrite was provided to all groups via their drinking water (140 ppm; approximately 40 mg/kg per day) to further evaluate NDELA formation in vivo under non-bolus dosing situations. Sample collection in the second trial was limited to blood, gastric contents and liver. Preliminary experiments to establish peak blood NDELA concentrations and dose-response of NDELA formation in blood and gastric contents of mice (method validation) were undertaken using oral gavage dosing of DEA plus sodium nitrite. Urine, blood and gastric contents were analyzed for NDELA and blood was also analyzed for DEA. Livers were excised in the second trial and the hepatic concentrations of a number of phospholipids, choline and cholinemetabolites were determined.
2.4. Dosing In both trials, animals were dosed at a constant level of 160 mg/kg per day based upon the most recent body weights. Oral gavage dosages were prepared in distilled water at a dosing volume of
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10 ml/kg and were delivered using a 1 cc tuberculin syringe equipped with a disposable gavage needle. Dermal application sites consisted of a roughly 4 cm2 area running from the interscapular region several cm posterior of this. The area was clipped free of fur, depilated using a commercial depilatory, rinsed with a wet paper towel and blotted dry prior to initiation of dosing and subsequently on an as-needed basis. Care was taken during the preparation of the site to avoid irritation of the skin. Dermal administration mimicked the NTP (1997) bioassay of DEA in that a 90 mg/ml concentration in ethanol was administered at a volume of 1.78 ml/kg. Mice were dosed by spreading a known volume of test material about the prepared target site using a micropipetter equipped with a blunted tip. In the first trial, access to the application site was prevented by use of an ADVANCED CURAD® AQUA-PROTECT™ bandage (38 mm× 38 mm) placed over the dosing site. In the second trial, a Teflon® collar consisting of a 20 mm band of flexible Teflon, as described by Wahle et al. (1998) was fitted about the necks of mice to prevent animals from grooming. Another group was allowed access to the application site during normal grooming activities. A group of six untreated, but clipped and depilated, mice served as controls in each trial. In the second trial, sodium nitrite spiked drinking water was changed every 3 days during the dosing period.
2.5. Sample collection In the first trial, urine was collected on test days 5–7 and 12–14 by placing mice in a Roth-type glass metabolism cage. Urine was collected on dry ice and was acidified to prevent further NDELA production. In both trials, mice were sacrificed approximately 1–2 h after the final dose. Mice were anesthetized with methoxyflurane and exsanguinated by cardiac puncture. In the second trial, livers and stomachs of all mice were then excised, stomachs were split open and gastric contents recovered. Gastric contents, liver and blood samples were then weighed, frozen in liquid nitrogen and stored at − 80°C. Blood was analyzed for DEA. Urine, blood and gastric contents were
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analyzed for NDELA concentration and liver was analyzed for PtdCho, PtdEtn, choline, SM, PCho, and GPCho.
2.6. DEA and NDELA analyses DEA was determined in blood and NDELA was determined in urine, gastric contents and blood of all mice using gas chromatographymass spectrometry (GC-MS). Samples for DEA or NDELA analysis were weighed and spiked with a known amount of a D8-DEA and D8NDELA internal standard, respectively, to ensure accurate quantitation. DEA whole blood samples were then pH-adjusted with 2.5 N KOH and derivatized with pentafluorobenzoyl chloride, extracted into toluene and analyzed. NDELA urine samples were acidified with 1 N HCl while gastric contents and blood samples were fortified with 20% NaCl and ammonium carbonate ( 25 mg). All NDELA samples were subsequently extracted into ethyl acetate, concentrated to dryness with N2, and derivitized with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) in toluene. Derivatized DEA and NDELA were then analyzed using GC-MS to ensure structural confirmation of DEA and NDELA present in the appropriate samples. Separations were achieved on a DB5ms capillary column (J&W Scientific, Folsom, CA) and the mass spectrometer was operated in the negative ion/chemical ionization mode (CH4) monitoring ions 332 (NDELA) and 340 (D8NDELA), or 687 (DEA) and 695 (D8-DEA). Analytical standards were prepared in control matrix. Limits of detection (LOD) of NDELA were determined to be 324 ng/g in urine, 1.8 ng/g in gastric contents, and 5 and 1.5 ng/g in blood in the first and second trials, respectively.
Dyer, 1959), spiked with appropriate [14C]methyl and [2H]methyl labeled internal standards to permit peak selection and calculation of recovery efficiency, and separated into organic and aqueous phases. Hydrophilic choline metabolites were then separated by high performance liquid chromatography and quantitated using gas chromatography-mass spectroscopy. PtdCho, PtdEtn and SM were analyzed using the methods outlined by Svanborg and Svennerholm (1961) and Yen et al. (1999). Briefly, following extraction, hydrophobic metabolites were separated using thin layer chromatography (TLC), recovered by scraping appropriate bands and quantitated along with authentic phospholipid standards using a phosphorous assay.
2.8. Clinical chemistry (second trial) Serum was harvested from collected blood samples and the activies of alanine and aspartate aminotransferases (ALT and AST, respectively) were determined using a Hitachi Model 914 Automatic Analyzer (Boehringer Mannheim, Indianapolis, IN).
2.9. Statistics All measurable parameters were first evaluated for homogeneity of variance (a= 0.01) followed by a one-way analysis of variance (ANOVA) with dose as the factor (Winer, 1971). If the dose effect was significant, a Tukey’s HSD test was conducted at an a= 0.05 (Kramer, 1956).
3. Results
3.1. NDELA analyses 2.7. Choline and phospholipid analyses (second trial) Choline and its metabolites PCho and GPCho were analyzed using the methods outlined by Zeisel et al. (1995) and Pomfret et al. (1989). Briefly, liver samples were extracted (Bligh and
No NDELA was identified in the urine or blood of mice from any treatment group in the absence of supplemental nitrite intake (first trial) or in blood or gastric contents when supplemental nitrite was administered (second trial).
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3.2. Blood DEA analyses The levels of DEA attained in the blood of mice at 1 –1.5 h post-dosing on the final day of the dosing period are shown in Fig. 1. Blood levels ranged from 5 to 7.7 mg/g in a route/method dependent manner. Dermal absorption of DEA administered in an ethanol vehicle over the 2week dosing period resulted in blood levels approximately 65% that obtained from administration of DEA via oral gavage. A trace amount of DEA was also found in the blood of control mice.
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2a–c, choline, PCho and GPCho decreased significantly relative to controls by as much as 64, 84 and 70%, respectively. The smallest decreases occurred in dermal (collared mice) treated mice and the largest in mice administered DEA via oral gavage. PtdCho and PtdEtn levels from the same DEA-treated groups of animals were generally decreased about 20% with the exception of a minimal decrease in PtdCho levels of mice administered DEA via dermal administration (Fig. 2d– e). In contrast, SM levels were increased in treated mice relative to controls (Fig. 2f).
3.3. Choline/phospholipid analyses
3.4. Clinical appearance and serum enzyme le6els
Results of analyses for hepatic PCho, choline, GPCho, PtdCho, PtdEtn, and SM are shown in Fig. 2a–f. A pronounced decrease in choline and its metabolites was observed in the livers of all DEA-treated groups of mice. As shown in Fig.
No clinical signs of toxicity were observed in mice treated with DEA and sodium nitrite (second trial). In addition, ALT and AST levels in DEA treated mice were similar to those of controls (data not shown).
Fig. 1. Blood levels of diethanolamine (DEA) 1–2 h post-dosing in B6C3F1 mice administered 0 (controls) or 160 mg/kg per day DEA via dermal application without access to the dosing site (dermal), dermal application with access during grooming (dermal and oral) or oral gavage (oral). All animals also received approximately 40 mg/kg per day sodium nitrite via their drinking water. [Means and standard deviations (S.D.) of 5 mice/group; * denotes significantly different from controls and different letters denote statistical differences between administration methods; Tukey’s HSD test at an a =0.05.]
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Fig. 2. Hepatic levels of phospholipid and choline-related compounds in B6C3F1 mice administered 0 (controls) or 160 mg/kg per day diethanolamine (DEA) via dermal application without access to the dosing site (Dermal), dermal application with access during grooming (dermal and oral) or oral gavage (oral). All animals also received approximately 40 mg/kg per day sodium nitrite via their drinking water. [Means and standard deviations (S.D.) of 5 mice/group; * denotes significantly different from controls and different letters denote statistical differences between administration methods; Tukey’s HSD test at an a =0.05.]
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Fig. 2. (Continued)
4. Discussion The findings of this study revealed several significant factors impacting on the interpretation of the dermal bioassay of DEA in rodents. These included: Evidence of dermal absorption of DEA in repeatedly dosed mice and the impact of grooming upon absorption of an administered dermal dose of DEA. Lack of significant levels of nitrosamine (NDELA) formation in vivo in mice dosed with tumorigenic dosages of DEA (even orally). Depletion of choline-containing compounds in liver of DEA-treated mice. The comparison of blood levels of DEA obtained in mice administered DEA via strictly dermal means (collared mice) versus oral gavage (Fig. 1) indicates the absorption of dermally applied DEA in mice. Despite the fact that blood data were obtained at a single time point and potential differences between the pharmacokinetics of dermally applied versus oral bolus dosed DEA, blood levels from the former were roughly
65% that obtained from the latter means of administration. This is consistent with the findings of Mathews et al. (1997), who reported that up to 60% of an administered dose of DEA in an ethanol vehicle was absorbed by B6C3F1 mice. The impact of allowing the animals access to the site of application was also apparent as a 35% increase in blood levels obtained in mice allowed access (dermal+ oral) versus those prevented access (wearing collars). Initial analyses of NDELA spiked-urine, blood and gastric ingesta revealed that the latter two matrices provided a more sensitive indicator of nitrosamine formation in vivo than measurements in mouse urine. LODs as low as 1.5–1.8 ng/g in blood and ingesta were attained. Preliminary experiments also established optimal time post-dosing when peak blood levels and measurable NDELA levels in blood and gastric contents would have been expected to occur. No NDELA was found in the urine or blood of male B6C3F1 mice repeatedly administered a carcinogenic dosage of 160 mg/kg per day DEA via dermal application (grooming of application site prevented), a combined dermal and oral (groom-
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ing allowed), or oral gavage in the absence of supplemental nitrite in the first trial. Nitrate consumption via feed and drinking water, estimated to be approximately 5 mg/kg per day, and its presumed conversion to nitrite by gut flora did not support sufficient NDELA synthesis to be detected at the LODs of 324 and 5 ng/g for urine and blood, respectively (first trial). Even upon the addition of supplemental nitrite (170 mg/ml NaNO2) to drinking water, no NDELA was detected in blood or gastric contents of mice repeatedly administered 160 mg/kg per day DEA via oral gavage or skin painting at an LOD of 1.5 – 1.8 ng/g (second trial). Thus, the non-bolus administration of sodium nitrite to mice via drinking water resulted in \2000-fold lower NDELA blood levels than when gavage dosing was employed in preliminary experiments (4.7 mg/ml). Further evidence for the impact of bolus dosing upon NDELA formation comes from Konishi et al. (1986) who recovered 823 mg NDELA in a gastric rinse of rats administered 58 mg/kg DEA and 153 mg/kg sodium nitrite via oral gavage. In contrast, the theoretical maximum total NDELA possible in the ingesta of treated mice in the present study was only 0.35 mg. These data suggest that generation of even low levels of NDELA require relatively large, bolus, dosages of DEA and nitrosating compound(s). As noted, alterations in choline metabolism may have far reaching effects upon the homeostasis of a number of cellular functions including; membrane function, rates of cell proliferation and apoptosis, activity of protein kinase C, and maintenance methylation of DNA (reviewed by Zeisel and Blusztajan, 1994). PCho represents the most labile choline storage pool in rodents while GPCho likely represents a metabolite formed during PtdCho-dependent choline generation (Zeisel et al., 1995). The smaller decreases in PtdCho levels relative to choline and PCho, decrease in the PtdCho metabolite GPCho, and DEA-induced decreases in the rate of PtdCho synthesis reported by Barbee and Hartung (1979) in liver, albeit of rats, suggest that membrane PtdCho is preserved, despite significant depletion of choline precursors by slowing its hydrolysis to GPCho. Thus, a higher demand may be placed upon dietary sources of choline.
The small decreases in hepatic PtdEtn observed in DEA-treated mice (Fig. 2e) likely reflected a portion of DEA incorporated during PtdEtn synthesis (Barbee and Hartung, 1979; Mathews et al., 1995). Again, Barbee and Hartung (1979) had also observed significant decreases in PtdEtn synthesis rates in DEA treated rats. Less clear was the elevation in SM levels directly correlated to blood DEA levels (Fig. 2f). However, this is consistent with the finding by Mathews et al. (1995) that DEA-altered ceramides, and in particular SM, represent the metabolic pool containing a majority of incorporated DEA. Methods utilized to separate and quantitate SM, TLC followed by total phosphorous determination, would not have differentiated between native SM and DEAmodified SM. Ironically, it was reported that choline deficiency was not a factor in the mouse bioassay as fatty liver, a standard histopathological response to dietary choline-deficiency, was not observed in the study (NTP, 1997). The present results indicate that DEA causes this deficiency and demonstrate that biochemical changes can occur without producing fatty liver. Subsequent changes associated with choline deficiency such as hypomethylation of DNA, increased oxidative stress and enhanced S-phase DNA synthesis, which are inturn associated with tumor formation in rodents (reviewed by Goodman et al., 1991), could have accounted for the observed bioassay results. Possible consequences of DEA-induced choline deficiency in mice related to tumorigenesis are presently under study.
Acknowledgements The authors wish to acknowledge the vision and support of Drs H.C. Pitot (Univ. Wisconsin) and L.D. Lehman-McKeeman (Procter & Gamble), Ms L. McFadden (statistics), Mr R. Papciak (Huntsman Chemical Co.), Dr H-W. Leung (Union Carbide) and Dr M. Kayser (formerly of BASF Corp.), and the financial support of the Chemical Manufacturers Association (Alkanolamines Panel) and Cosmetic, Toiletry & Fragrance Association in the design and conduct of
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this work. Dr Zeisel is supported by a grant from NIH (AG 09525).
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