Species Differences in the Induction of Hepatocellular DNA Synthesis by Diethanolamine
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《毒物学科学杂志》
Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202
ABSTRACT
Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0–500 μg/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 μg/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.
Key Words: diethanolamine; choline depletion; DNA synthesis; hepatocytes.
INTRODUCTION
Diethanolamine is an alkanolamine used widely in industry and is found in a variety of consumer products. Human exposure to diethanolamine is possible through dermal contact with personal care products that contain this compound. Chronic dermal exposure to diethanolamine (40, 80, or 160 mg/kg diethanolamine in 1.8 ml of 95% ethanol) increased both the incidence and multiplicity of hepatocellular adenoma and carcinoma in male and female B6C3F1 mice, whereas no increase in hepatocellular neoplasia was seen in F344 rats exposed to 8, 16, or 32 mg/kg diethanolamine in 0.6 ml of 95% ethanol (National Toxicology Program, 1999). No oral carcinogenicity studies for diethanolamine have been performed in rodents. The potential mutagenicity and carcinogenicity of diethanolamine has been evaluated (Knaak et al., 1997; National Toxicology Program, 1999). Genotoxicity studies performed in several test systems including Salmonella typhimurium, L5178Y mouse lymphoma cells, and Chinese hamster ovary cells showed no evidence of genotoxicity (Knaak et al., 1997; National Toxicology Program, 1999). These findings are suggestive of an epigenetic mode of carcinogenic action for diethanolamine in B6C3F1 mice.
Diethanolamine inhibits choline uptake in cells and causes a pronounced dose-related decrease in hepatic choline pools in treated mice (Lehman-McKeeman and Gamsky, 1999; Lehman-McKeeman et al., 2002; Stott et al., 2000). Choline deficiency has been shown to disrupt cellular growth and division (Terce et al., 1994; Yen et al., 1999), alter hepatic and renal function (Kratzing et al., 1972; Zeisel and Blusztajn, 1994), and cause spontaneous carcinogenesis in rodents (Newberne et al., 1982; Zeisel, 1996). These observations have led to the hypothesis that the mode of action for diethanolamine-induced tumors in the mouse liver occurs through depletion of cellular choline.
The induction of DNA synthesis and modulation of cell growth by chemical carcinogens is an important component of the carcinogenesis process for both genotoxic and nongenotoxic carcinogens (Butterworth, 1990; Pitot et al., 1981). Cell proliferation can result in an increase in spontaneous mutations acquired during DNA synthesis and/or the selective clonal expansion of previously initiated cells (Klaunig, 1993; Schulte-Hermann 1987), or may alter methylation of the genome and facilitate clonal expansion of initiated cells via changes in gene expression that silence tumor suppressor genes or increase expression of oncogenes in the liver, either of which may result in the formation of hepatic focal lesions.
An increase in cell growth can occur through increases in mitosis and DNA synthesis and/or a reduction of apoptosis (Goldsworthy et al., 1993; Klaunig, et al., 2000). Thus, the examination of the effect of diethanolamine on induction of cell proliferation is important to further our understanding of the mechanism(s) for diethanolamine carcinogenicity. Previously, increased cell proliferation in liver from B6C3F1 mice treated with diethanolamine was seen following 1 week of exposure and persisted through the 13 weeks examined (Mellert et al., 2004). These studies further established a threshold level of 10 mg diethanolamine/kg body weight for diethanolamine-induced DNA synthesis, further supporting a nongenotoxic mode of action involving the induction of cell proliferation.
To study the mode of action for diethanolamine-induced liver neoplasia and the plausibility that choline depletion is involved in the mode of action, the present studies examined DNA synthesis and gene expression changes in mouse and rat hepatocytes. The effect of choline depletion and the effects of choline supplementation on diethanolamine-induced DNA synthesis was examined. In addition, for comparative purposes, DNA synthesis following diethanolamine treatment was examined in human hepatocytes.
MATERIALS AND METHODS
Animals.
Male B6C3F1 mice and F344 rats (6–8 weeks of age) were purchased from Harlan Sprague-Dawley. Animals were housed and maintained in an AAALAC approved facility at Indiana University. Animals were maintained in accordance with the NIH guide for the care and use of laboratory animals.
Isolation and culture of hepatocytes.
Cryopreserved human hepatocytes were obtained from In Vitro Technologies (Baltimore, MD). All protocols for use of human hepatocytes were reviewed and approved by the Institutional Review Board of Indiana University prior to initiation of this study (IU IRB#0109–54). Hepatocytes were thawed and suspended in DMEM-F12 medium. Cells were then centrifuged (1x at 50 x g, 5 min), and viability was determined by trypan blue exclusion, and was routinely greater than 88%. Cells were plated on collagen-coated dishes (0.35 x 106 cells, 35 mm) in DMEM-F12 medium containing insulin (5 μg/ml), gentamicin sulfate (50 mg/ml), dexamethasone (0.8 μg/ml), and 10% fetal bovine serum. Mouse and rat hepatocytes were isolated by 2-step in situ collagenase perfusion as previously described (Klaunig et al., 1981). Following perfusion, hepatocytes were filtered and centrifuged (2x, 300 x g, 3 min). Viability was determined by trypan blue exclusion, and cells used when viability exceeded 90%. Cells were plated (1 x 106 cells, 60-mm dishes) in DMEM-F12 medium containing insulin (5 μg/ml), gentamycin sulfate (50 mg/ml), dexamethasone (0.8 μg/ml), and 5% fetal bovine serum. All hepatocyte cultures were maintained at 5% CO2, 37°C, and 95% humidity, and media was changed after 4 h. Treatments were added to culture dishes following overnight incubation.
Assessment of cytolethality.
Initial studies were conducted in primary hepatocytes isolated from mouse liver to establish noncytolethal concentration ranges for diethanolamine. Cytolethality was determined in primary cultured hepatocytes by the release of lactate dehydrogenase (LDH) into cell culture medium. Hepatocytes were treated with diethanolamine at concentrations ranging from 0 to 2000 μg/ml, and cytolethality was determined following 24 h of culture.
Quantitation of DNA synthesis.
Replicative DNA synthesis was measured according to the method of James and Roberts (1996). BrdU (20 mM final concentration) was added to cell cultures and incubated for the last 16 h of culture. Cell cultures were then washed and fixed with methanol. Incorporated BrdU was visualized using an anti-BrdU antibody followed by a peroxidase linked secondary antibody and a DAB substrate. Replicative DNA synthesis was measured by scoring the percentage of BrdU positive nuclei in a minimum of 1000 hepatocytes. Results were obtained from replicate cultures in four independent experiments.
Gene expression analysis.
cDNA microarray technology was used to examine the effects of diethanolamine treatment in isolated mouse and rat hepatocytes. Cells were treated for 24 h with either diethanolamine or medium containing reduced choline, and total mRNA was isolated for the analysis of gene expression using commercially available microarrays containing representative genes associated with pathways specific for apoptosis or cell proliferation (SuperArray, Frederick, MD). Membranes were quantified using TotalLab Imagae analysis software (Nonlinear USA Inc., Durham, NC). Data are expressed as fold-changes in gene expression compared to control hepatocytes.
Statistical Analysis.
The data was analyzed by ANOVA followed by a Dunnett's two-tailed test. For all studies, treatment groups were considered significantly different from control values when p < 0.05.
RESULTS
Diethanolamine cytotoxicity (LDH release) was examined in mouse, rat, and human hepatocytes to determine the highest noncytolethal concentration of diethanolamine to be used in subsequent studies evaluating DNA synthesis and gene expression changes. The cytolethality of diethanolamine was similar in hepatocytes from the three species examined. Results demonstrated that diethanolamine induced significant increases in LDH release at concentrations of 750 μg/ml and above in mouse, rat, and human hepatocytes (data not shown).
DNA synthesis was increased in diethanolamine-treated mouse hepatocytes at concentrations of 10 μg/ml diethanolamine and higher, producing an increase that ranged from a 1.2- to 2.5-fold over control (Fig. 1). Diethanolamine resulted in similar increases in DNA synthesis in rat hepatocytes, producing an approximate 2-fold increase in DNA synthesis at 10 μg/ml, which increased to >3-fold over control at 250–750 μg/ml diethanolamine (Fig. 2). In experiments examining DNA synthesis in mouse and rat hepatocytes, phenobarbital served as a positive control and produced an approximate 1.6-fold increase in DNA synthesis in mouse hepatocytes and a 2.3-fold increase in rat hepatocytes, compared to control (Figs. 1 and 2). In human hepatocytes, diethanolamine did not increase DNA synthesis at concentrations up to 750 μg/ml of diethanolamine, the highest noncytolethal concentration examined (Fig. 3). Importantly, treatment with epidermal growth factor (EGF) resulted in a 2-fold increase in DNA synthesis in all preparations of human hepatocytes evaluated (Fig. 3). These results demonstrate that the human hepatocytes were responsive to an established growth stimuli.
It has been speculated that the induction of hepatic neoplasia by diethanolamine is due to inhibition of choline uptake and subsequent cellular depletion of choline (Lehman-McKeeman and Gamsky, 1999; Lehman-McKeeman et al., 2002). Therefore, studies were performed to evaluate the effect of choline depletion on the induction of DNA synthesis in mouse, rat, and human hepatocytes. Reduced choline in cell culture medium (from 1/10 to 0 choline in culture medium) for 24 h was noncytolethal in mouse, rat, and human hepatocytes (data not shown). In mouse hepatocytes, choline depletion increased DNA synthesis by approximately 1.5-fold over control after 24 h (Fig. 4), and was significantly increased when the choline concentration was lowered to 1/50 of the normal choline level in culture medium. Increases in DNA synthesis were also seen in rat hepatocytes cultured with reduced choline in culture medium (Fig. 5). In rat hepatocytes, all levels of choline reduction in culture medium (1/10 the normal content and lower) produced 2- to 2.5-fold increases in DNA synthesis (Fig. 5). Similar to the effect of diethanolamine in human hepatocytes, reduction of choline concentration in culture medium did not increase DNA synthesis in human hepatocytes, while again, the cell cultures were responsive (2.5-fold increase in DNA synthesis) to EGF (Fig. 6).
Supplementing cell culture medium with choline (2- to 50-fold excess choline; 18–449 mg choline/l culture medium) for 24 h did not induce cytolethality in either mouse or rat hepatocytes (data not shown). However, choline supplementation reduced basal levels of DNA synthesis in mouse hepatocytes compared to control (Fig. 7). An apparent dose-related decrease in DNA synthesis was observed that was significantly lower than control values at choline concentrations of 5x (44.9 mg choline/l medium) normal culture medium and higher in culture medium in mouse hepatocytes (Fig. 7). In rat hepatocytes, choline supplementation produced only slight decreases in basal levels of DNA synthesis regardless of concentration of choline supplemented (Fig. 8).
Cotreatment of mouse hepatocytes with diethanolamine and choline showed a concentration-related reduction of diethanolamine-induced DNA synthesis by choline supplementation (Fig. 9). A significant reduction in diethanolamine-induced DNA synthesis was seen following supplementation with all choline concentrations (2- to 50-fold), whereas a significant reduction in both the basal level of DNA synthesis and diethanolamine-induced DNA synthesis was seen following supplementation with 5-fold excess choline and higher in mouse hepatocytes (Fig. 9). In rat hepatocytes, choline supplementation at levels at least twice the amount of choline in culture medium reduced diethanolamine-induced DNA synthesis to that of control levels (Fig. 10). However, as seen with choline supplementation on basal DNA synthesis in rat hepatocytes, choline supplementation in the presence of diethanolamine did not reduce DNA synthesis below basal levels (Fig. 10).
Studies examining alteration of gene expression by diethanolamine and choline depletion were performed in mouse and rat hepatocytes treated for 24 h with 500 μg/ml diethanolamine or culture medium containing 1/100 the normal choline content. (treatments that increased DNA synthesis). The results presented in Table 1 are a summation of gene expression changes that were significantly different between either diethanolamine or choline depletion compared to gene expression in control hepatocytes cultures. In these studies, diethanolamine altered expression of genes involved in cell growth regulation, exhibiting changes in expression of genes involved in both induction of DNA synthesis/cell cycle and suppression of apoptosis (Table 1). In particular, several cell cycle genes were upregulated, while genes involved in the p53-mediated apoptotic pathway and the caspase family were decreased in both mouse and rat hepatocytes (Table 1). Interestingly, reduced choline in cell culture medium resulted in changes in gene expression similar to those observed following diethanolamine treatment in both mouse and rat hepatocytes (Table 1).
DISCUSSION
Chronic dermal exposure to diethanolamine resulted in an increased incidence of liver tumors (adenomas, carcinomas, and hepatoblastomas) in B6C3F1 mice at doses of 40 mg/kg diethanolamine per day (National Toxicology Program, 1997). Diethanolamine was negative for genotoxicity or mutagenicity in several assays (Knaak et al., 1997) and appears to produce hepatic neoplasia in B6C3F1 mice via an epigenetic mechanism.
A substantial body of evidence is emerging suggesting a role for inhibition of cellular choline uptake in diethanolamine carcinogenicity (Lehman-McKeeman et al., 2002; Rogers et al., 1987; Stott et al., 2000; Zeisel, 1996). Based on the results presented in the current study and other published data, a mode of action for diethanolamine carcinogenicity, based on cellular choline depletion, has been proposed (Fig. 11). This mode of action identifies the following key steps. (1) Diethanolamine inhibits choline uptake, which leads to cellular choline depletion. (2) Decreased choline levels then reduce the pool of 1-carbon donor groups, which lowers the potential for methylation reactions. (3) Alteration of DNA methylation affects gene expression, leading to altered expression of genes involved in cell growth regulation, (4) which in turn promotes the growth of preexisting (spontaneously) initiated preneoplastic hepatocytes in B6C3F1 mouse liver and ultimately produces hepatic neoplasia (Fig. 11).
Several lines of experimental evidence support this proposed mode of action. Diethanolamine is known to disrupt intracellular choline homeostasis by inhibiting choline uptake (Lehman-McKeeman and Gamsky, 1999, 2000). Important to the proposed mechanism, inhibition of choline uptake and decreased phosphocholine synthesis were reversed by removing diethanolamine from cell culture medium and prevented by supplementing the culture medium with choline (Lehman-McKeeman and Gamsky, 1999, 2000). Furthermore, diethanolamine-induced morphological transformation in Syrian Hamster Embryo cells was prevented by choline supplementation (Lehman-McKeeman and Gamsky, 2000). In vivo, hepatic levels of choline metabolites and S-adenosyl methionine (SAM) were decreased in B6C3F1 mice dosed with diethanolamine, while no changes were observed in F344 rat liver (Lehman-McKeeman et al., 2002; Stott et al. 2000).
Choline depletion has been shown to function as a tumor promoter in rodents, and choline deficiency by itself is a nutritional deficiency considered carcinogenic in rodents (DeCarmargo et al., 1985; Newberne et al., 1982; Rogers et al., 1987). In vivo, choline that is not phosphorylated is oxidized to betaine, a product that serves as a methyl donor in a reaction that converts homocysteine to methionine. Methionine can then be converted to SAM, a methyl donor for many enzymatic reactions (Zeisel and Blusztajn, 1994). Since diethanolamine interferes with cellular choline uptake and results in choline depletion, diethanolamine treatment would be expected to reduce SAM levels and lower the potential for DNA methylation. Hypomethylation of DNA is considered an epigenetic mechanism of carcinogenesis and has been shown to activate a number of genes including oncogenes (Eden et al., 2003; Goodman and Watson, 2002). DNA hypomethylation was observed in rat liver after 7 days on a choline- or lipotrope-deficient diet (Locher et al., 1986; Wainfen et al., 1989). Thus, hypomethylation of critical target genes as a result of reduced availability of SAM could be a critical factor in the carcinogenic response observed in mice following diethanolamine exposure. Currently, studies are ongoing to determine the effect of diethanolamine on methylation status in mouse and rat hepatocytes.
The induction of DNA synthesis is an obligate event for the development of neoplasia. Choline deficiency is known to induce liver cell proliferation (Abanobi et al., 1982; Counts et al., 1996; Zeisel, 1996). In the current studies, DNA synthesis was increased 1.8- to 3.2-fold over control in mouse hepatocytes following exposure to diethanolamine at and above 10 μg/ml. Incubation of mouse hepatocytes in medium containing reduced choline concentrations (1/10 to 1/100 of normal medium; 0.898 mg/l to 0.0898 mg/l) for 24 h produced similar increases (1.4- to 2.4-fold) in DNA synthesis over control, whereas incubation of cells in medium containing 10-fold higher choline concentrations produced a 50% reduction in DNA synthesis compared to control groups. These results demonstrate that modulation of cellular choline levels modulates DNA synthesis in rodent hepatocytes.
In studies examining species selectivity of diethanolamine, increases in DNA synthesis were observed in mouse and rat, but not human hepatocytes following treatment with diethanolamine. Furthermore, incubation of hepatocytes in medium containing reduced choline increased DNA synthesis 1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively, while no increase was observed in human hepatocytes. Choline supplementation also reduced diethanolamine-induced DNA synthesis to control levels or below in mouse and rat hepatocytes. These results indicate that the induction of in vitro DNA synthesis by diethanolamine in the liver is associated with choline depletion, and is species specific. Similarly, diethanolamine elicited a dose-related increase in DNA synthesis in B6C3F1 mouse liver following dermal application of diethanolamine (160 mg/kg/d) for 1 and 13 weeks of exposure (Mellert et al., 2004). In mice dosed with diethanolamine for 1 week then allowed 3 weeks of recovery (no diethanolamine), hepatic labeling indices returned from 3-fold increase over control to control values, indicating that the proliferative effect of diethanolamine was reversible (Mellert et al., 2004). These findings provide further support that diethanolamine induces neoplasia through epigenetic mechanisms and functions at the promotion stage of the carcinogenesis process.
The present studies also examined the effects of diethanolamine and choline depletion on expression of genes involved in cell growth regulation in mouse and rat hepatocytes using cDNA microarrays. mRNA from mouse and rat hepatocytes treated with diethanolamine or choline depletion showed similar patterns of gene expression, altering several genes involved in pathways associated with cell cycle progression or suppression of apoptosis. In particular, p53 expression was decreased. This gene functions in several pathways including p21cip, which acts to inhibit cyclin D expression. Cyclin D acts on the Rb gene, in concert with E2F transcription factor, to drive the cell cycle. In the present experiments, both p53 and p21cip expression were decreased, while expression of Cyclin D1, Cyclin E, and E2F were increased. Therefore, the reduced p21cip block, coupled with increased cyclin and E2F expression, is permissive for entry into the cell cycle. Expression of p19, a gene encoding a protein that functions to inhibit cyclin D expression, was also decreased. In addition, caspase 3, bad, and bax expression exhibited a reduced expression. Reduction in expression of these pro-apoptotic genes would also be expected to reduce the number of cells undergoing apoptosis. Overall, the gene expression profile observed following treatment with diethanolamine or choline depletion treatment demonstrated an alteration of genes involved in pathways leading to a reduction in apoptosis as well as a progression into the cell cycle.
Chronic dermal exposure to diethanolamine resulted in a significant increase in liver neoplasia in the liver of treated mice but not the rat (National Toxicology Program, 1999). To date, no oral carcinogenicity studies with diethanolamine have been performed in rodents. In the present studies, diethanolamine altered expression of genes involved in cell growth regulation and induced DNA synthesis in rat hepatocytes. Following dermal application of diethanolamine in vivo, it has been shown that absorption of diethanolamine is greater in mice (25–60%) than in rats (3–16%) and results in approximately 3-fold higher blood and tissue concentrations of diethanolamine in mice compared to rats (Mathews et al., 1995, 1997; Mendrala et al., 2001). In the in vitro system used in the current studies, equimolar concentrations of diethanolamine were delivered to mouse and rat hepatocytes, whereas in vivo, due to differences in dermal absorption between species and differences in sensitivities to diethanolamine-induced skin irritation, blood and tissue concentrations of diethanolamine are lower in rats than in the mouse (Matthews et al., 1997). Thus, the concentrations of diethanolamine administered to rat hepatocytes were relatively high and were able to disrupt choline status and subsequently induce gene expression changes and DNA synthesis.
With respect to human risk from exposure to diethanolamine, the proposed mode of action for diethanolamine-induced choline deficiency is qualitatively applicable to humans. However, marked species differences in susceptibility to choline deficiency exist, with mice and rats being more susceptible than other species including humans, which are generally considered refractory to choline deficiency due to low levels of choline oxidase activity (Hoffbauer and Zaki, 1965; Sidransky and Farber, 1960; Zeisel and Blusztajn, 1994). Furthermore, diethanolamine absorption is considerably lower in humans compared to rodents. The differences in the absorptive properties of diethanolamine between species also correlates with the susceptibility to choline deficiency, with mice and rats being more susceptible than humans (Sun et al., 1996).
Several other hypotheses have been proposed for diethanolamine-induced hepatic neoplasia. Diethanolamine is a secondary amine that can undergo nitrosation to form a mutagenic nitrosamine, N-nitrosodiethanolamine. While N-nitrosodiethanolamine induced liver tumors in rats at doses of 2 mg/kg per day, this nitrosamine is not produced in measurable amounts following administration of diethanolamine at carcinogenic doses, suggesting that diethanolamine carcinogenicity does not result from N-nitrosodiethanolamine production (ECETOC, 1990; Preussmann, et al., 1981; Stott et al., 2000; Yamamoto et al., 1995). Due to structural similarity to choline and ethanolamine, diethanolamine also disrupts phospholipid metabolism, membrane function, and synthesis of fatty acid second messengers. Alterations in phospholipid metabolism in rats treated with diethanolamine, as well as the incorporation of diethanolamine into phospholipids and subsequent alteration of phospholipids have been reported (Barbee and Hartung, 1979; Mathews et al., 1995). However, the causality of these events to diethanolamine carcinogenicity has not been demonstrated.
Other effects associated with choline deficiency include increased generation of free radicals and increased susceptibility to oxidative damage (Floyd et al., 2002; Hensley et al., 2000; Rushmore et al., 1984) which may induce DNA damage and/or alter gene expression. An evaluation of reactive oxygen species or other oxidative stress parameters by diethanolamine has not been performed, and whether this mechanism participates in the induction of hepatic neoplasia by diethanolamine requires additional study.
Overall, the present results and those of other published studies support that the mode of action for diethanolamine hepatocarcinogenicity in mouse liver involves cellular choline deficiency that is associated with hypomethylation of DNA, alteration of gene expression, and increased DNA synthesis. Coupled together, these events result in the promotion of initiated cell populations in mouse liver. Further, the lack of induction of DNA synthesis by diethanolamine in human hepatocytes suggests that, due to species differences, humans are refractory to the carcinogenic effects of diethanolamine.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
This work was supported in part, by the Alkanolamines Panel of the American Chemistry Council (LMK), and by NIH R01CA100908 (J.E.K.). Conflict of interest: none declared.
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ABSTRACT
Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0–500 μg/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 μg/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.
Key Words: diethanolamine; choline depletion; DNA synthesis; hepatocytes.
INTRODUCTION
Diethanolamine is an alkanolamine used widely in industry and is found in a variety of consumer products. Human exposure to diethanolamine is possible through dermal contact with personal care products that contain this compound. Chronic dermal exposure to diethanolamine (40, 80, or 160 mg/kg diethanolamine in 1.8 ml of 95% ethanol) increased both the incidence and multiplicity of hepatocellular adenoma and carcinoma in male and female B6C3F1 mice, whereas no increase in hepatocellular neoplasia was seen in F344 rats exposed to 8, 16, or 32 mg/kg diethanolamine in 0.6 ml of 95% ethanol (National Toxicology Program, 1999). No oral carcinogenicity studies for diethanolamine have been performed in rodents. The potential mutagenicity and carcinogenicity of diethanolamine has been evaluated (Knaak et al., 1997; National Toxicology Program, 1999). Genotoxicity studies performed in several test systems including Salmonella typhimurium, L5178Y mouse lymphoma cells, and Chinese hamster ovary cells showed no evidence of genotoxicity (Knaak et al., 1997; National Toxicology Program, 1999). These findings are suggestive of an epigenetic mode of carcinogenic action for diethanolamine in B6C3F1 mice.
Diethanolamine inhibits choline uptake in cells and causes a pronounced dose-related decrease in hepatic choline pools in treated mice (Lehman-McKeeman and Gamsky, 1999; Lehman-McKeeman et al., 2002; Stott et al., 2000). Choline deficiency has been shown to disrupt cellular growth and division (Terce et al., 1994; Yen et al., 1999), alter hepatic and renal function (Kratzing et al., 1972; Zeisel and Blusztajn, 1994), and cause spontaneous carcinogenesis in rodents (Newberne et al., 1982; Zeisel, 1996). These observations have led to the hypothesis that the mode of action for diethanolamine-induced tumors in the mouse liver occurs through depletion of cellular choline.
The induction of DNA synthesis and modulation of cell growth by chemical carcinogens is an important component of the carcinogenesis process for both genotoxic and nongenotoxic carcinogens (Butterworth, 1990; Pitot et al., 1981). Cell proliferation can result in an increase in spontaneous mutations acquired during DNA synthesis and/or the selective clonal expansion of previously initiated cells (Klaunig, 1993; Schulte-Hermann 1987), or may alter methylation of the genome and facilitate clonal expansion of initiated cells via changes in gene expression that silence tumor suppressor genes or increase expression of oncogenes in the liver, either of which may result in the formation of hepatic focal lesions.
An increase in cell growth can occur through increases in mitosis and DNA synthesis and/or a reduction of apoptosis (Goldsworthy et al., 1993; Klaunig, et al., 2000). Thus, the examination of the effect of diethanolamine on induction of cell proliferation is important to further our understanding of the mechanism(s) for diethanolamine carcinogenicity. Previously, increased cell proliferation in liver from B6C3F1 mice treated with diethanolamine was seen following 1 week of exposure and persisted through the 13 weeks examined (Mellert et al., 2004). These studies further established a threshold level of 10 mg diethanolamine/kg body weight for diethanolamine-induced DNA synthesis, further supporting a nongenotoxic mode of action involving the induction of cell proliferation.
To study the mode of action for diethanolamine-induced liver neoplasia and the plausibility that choline depletion is involved in the mode of action, the present studies examined DNA synthesis and gene expression changes in mouse and rat hepatocytes. The effect of choline depletion and the effects of choline supplementation on diethanolamine-induced DNA synthesis was examined. In addition, for comparative purposes, DNA synthesis following diethanolamine treatment was examined in human hepatocytes.
MATERIALS AND METHODS
Animals.
Male B6C3F1 mice and F344 rats (6–8 weeks of age) were purchased from Harlan Sprague-Dawley. Animals were housed and maintained in an AAALAC approved facility at Indiana University. Animals were maintained in accordance with the NIH guide for the care and use of laboratory animals.
Isolation and culture of hepatocytes.
Cryopreserved human hepatocytes were obtained from In Vitro Technologies (Baltimore, MD). All protocols for use of human hepatocytes were reviewed and approved by the Institutional Review Board of Indiana University prior to initiation of this study (IU IRB#0109–54). Hepatocytes were thawed and suspended in DMEM-F12 medium. Cells were then centrifuged (1x at 50 x g, 5 min), and viability was determined by trypan blue exclusion, and was routinely greater than 88%. Cells were plated on collagen-coated dishes (0.35 x 106 cells, 35 mm) in DMEM-F12 medium containing insulin (5 μg/ml), gentamicin sulfate (50 mg/ml), dexamethasone (0.8 μg/ml), and 10% fetal bovine serum. Mouse and rat hepatocytes were isolated by 2-step in situ collagenase perfusion as previously described (Klaunig et al., 1981). Following perfusion, hepatocytes were filtered and centrifuged (2x, 300 x g, 3 min). Viability was determined by trypan blue exclusion, and cells used when viability exceeded 90%. Cells were plated (1 x 106 cells, 60-mm dishes) in DMEM-F12 medium containing insulin (5 μg/ml), gentamycin sulfate (50 mg/ml), dexamethasone (0.8 μg/ml), and 5% fetal bovine serum. All hepatocyte cultures were maintained at 5% CO2, 37°C, and 95% humidity, and media was changed after 4 h. Treatments were added to culture dishes following overnight incubation.
Assessment of cytolethality.
Initial studies were conducted in primary hepatocytes isolated from mouse liver to establish noncytolethal concentration ranges for diethanolamine. Cytolethality was determined in primary cultured hepatocytes by the release of lactate dehydrogenase (LDH) into cell culture medium. Hepatocytes were treated with diethanolamine at concentrations ranging from 0 to 2000 μg/ml, and cytolethality was determined following 24 h of culture.
Quantitation of DNA synthesis.
Replicative DNA synthesis was measured according to the method of James and Roberts (1996). BrdU (20 mM final concentration) was added to cell cultures and incubated for the last 16 h of culture. Cell cultures were then washed and fixed with methanol. Incorporated BrdU was visualized using an anti-BrdU antibody followed by a peroxidase linked secondary antibody and a DAB substrate. Replicative DNA synthesis was measured by scoring the percentage of BrdU positive nuclei in a minimum of 1000 hepatocytes. Results were obtained from replicate cultures in four independent experiments.
Gene expression analysis.
cDNA microarray technology was used to examine the effects of diethanolamine treatment in isolated mouse and rat hepatocytes. Cells were treated for 24 h with either diethanolamine or medium containing reduced choline, and total mRNA was isolated for the analysis of gene expression using commercially available microarrays containing representative genes associated with pathways specific for apoptosis or cell proliferation (SuperArray, Frederick, MD). Membranes were quantified using TotalLab Imagae analysis software (Nonlinear USA Inc., Durham, NC). Data are expressed as fold-changes in gene expression compared to control hepatocytes.
Statistical Analysis.
The data was analyzed by ANOVA followed by a Dunnett's two-tailed test. For all studies, treatment groups were considered significantly different from control values when p < 0.05.
RESULTS
Diethanolamine cytotoxicity (LDH release) was examined in mouse, rat, and human hepatocytes to determine the highest noncytolethal concentration of diethanolamine to be used in subsequent studies evaluating DNA synthesis and gene expression changes. The cytolethality of diethanolamine was similar in hepatocytes from the three species examined. Results demonstrated that diethanolamine induced significant increases in LDH release at concentrations of 750 μg/ml and above in mouse, rat, and human hepatocytes (data not shown).
DNA synthesis was increased in diethanolamine-treated mouse hepatocytes at concentrations of 10 μg/ml diethanolamine and higher, producing an increase that ranged from a 1.2- to 2.5-fold over control (Fig. 1). Diethanolamine resulted in similar increases in DNA synthesis in rat hepatocytes, producing an approximate 2-fold increase in DNA synthesis at 10 μg/ml, which increased to >3-fold over control at 250–750 μg/ml diethanolamine (Fig. 2). In experiments examining DNA synthesis in mouse and rat hepatocytes, phenobarbital served as a positive control and produced an approximate 1.6-fold increase in DNA synthesis in mouse hepatocytes and a 2.3-fold increase in rat hepatocytes, compared to control (Figs. 1 and 2). In human hepatocytes, diethanolamine did not increase DNA synthesis at concentrations up to 750 μg/ml of diethanolamine, the highest noncytolethal concentration examined (Fig. 3). Importantly, treatment with epidermal growth factor (EGF) resulted in a 2-fold increase in DNA synthesis in all preparations of human hepatocytes evaluated (Fig. 3). These results demonstrate that the human hepatocytes were responsive to an established growth stimuli.
It has been speculated that the induction of hepatic neoplasia by diethanolamine is due to inhibition of choline uptake and subsequent cellular depletion of choline (Lehman-McKeeman and Gamsky, 1999; Lehman-McKeeman et al., 2002). Therefore, studies were performed to evaluate the effect of choline depletion on the induction of DNA synthesis in mouse, rat, and human hepatocytes. Reduced choline in cell culture medium (from 1/10 to 0 choline in culture medium) for 24 h was noncytolethal in mouse, rat, and human hepatocytes (data not shown). In mouse hepatocytes, choline depletion increased DNA synthesis by approximately 1.5-fold over control after 24 h (Fig. 4), and was significantly increased when the choline concentration was lowered to 1/50 of the normal choline level in culture medium. Increases in DNA synthesis were also seen in rat hepatocytes cultured with reduced choline in culture medium (Fig. 5). In rat hepatocytes, all levels of choline reduction in culture medium (1/10 the normal content and lower) produced 2- to 2.5-fold increases in DNA synthesis (Fig. 5). Similar to the effect of diethanolamine in human hepatocytes, reduction of choline concentration in culture medium did not increase DNA synthesis in human hepatocytes, while again, the cell cultures were responsive (2.5-fold increase in DNA synthesis) to EGF (Fig. 6).
Supplementing cell culture medium with choline (2- to 50-fold excess choline; 18–449 mg choline/l culture medium) for 24 h did not induce cytolethality in either mouse or rat hepatocytes (data not shown). However, choline supplementation reduced basal levels of DNA synthesis in mouse hepatocytes compared to control (Fig. 7). An apparent dose-related decrease in DNA synthesis was observed that was significantly lower than control values at choline concentrations of 5x (44.9 mg choline/l medium) normal culture medium and higher in culture medium in mouse hepatocytes (Fig. 7). In rat hepatocytes, choline supplementation produced only slight decreases in basal levels of DNA synthesis regardless of concentration of choline supplemented (Fig. 8).
Cotreatment of mouse hepatocytes with diethanolamine and choline showed a concentration-related reduction of diethanolamine-induced DNA synthesis by choline supplementation (Fig. 9). A significant reduction in diethanolamine-induced DNA synthesis was seen following supplementation with all choline concentrations (2- to 50-fold), whereas a significant reduction in both the basal level of DNA synthesis and diethanolamine-induced DNA synthesis was seen following supplementation with 5-fold excess choline and higher in mouse hepatocytes (Fig. 9). In rat hepatocytes, choline supplementation at levels at least twice the amount of choline in culture medium reduced diethanolamine-induced DNA synthesis to that of control levels (Fig. 10). However, as seen with choline supplementation on basal DNA synthesis in rat hepatocytes, choline supplementation in the presence of diethanolamine did not reduce DNA synthesis below basal levels (Fig. 10).
Studies examining alteration of gene expression by diethanolamine and choline depletion were performed in mouse and rat hepatocytes treated for 24 h with 500 μg/ml diethanolamine or culture medium containing 1/100 the normal choline content. (treatments that increased DNA synthesis). The results presented in Table 1 are a summation of gene expression changes that were significantly different between either diethanolamine or choline depletion compared to gene expression in control hepatocytes cultures. In these studies, diethanolamine altered expression of genes involved in cell growth regulation, exhibiting changes in expression of genes involved in both induction of DNA synthesis/cell cycle and suppression of apoptosis (Table 1). In particular, several cell cycle genes were upregulated, while genes involved in the p53-mediated apoptotic pathway and the caspase family were decreased in both mouse and rat hepatocytes (Table 1). Interestingly, reduced choline in cell culture medium resulted in changes in gene expression similar to those observed following diethanolamine treatment in both mouse and rat hepatocytes (Table 1).
DISCUSSION
Chronic dermal exposure to diethanolamine resulted in an increased incidence of liver tumors (adenomas, carcinomas, and hepatoblastomas) in B6C3F1 mice at doses of 40 mg/kg diethanolamine per day (National Toxicology Program, 1997). Diethanolamine was negative for genotoxicity or mutagenicity in several assays (Knaak et al., 1997) and appears to produce hepatic neoplasia in B6C3F1 mice via an epigenetic mechanism.
A substantial body of evidence is emerging suggesting a role for inhibition of cellular choline uptake in diethanolamine carcinogenicity (Lehman-McKeeman et al., 2002; Rogers et al., 1987; Stott et al., 2000; Zeisel, 1996). Based on the results presented in the current study and other published data, a mode of action for diethanolamine carcinogenicity, based on cellular choline depletion, has been proposed (Fig. 11). This mode of action identifies the following key steps. (1) Diethanolamine inhibits choline uptake, which leads to cellular choline depletion. (2) Decreased choline levels then reduce the pool of 1-carbon donor groups, which lowers the potential for methylation reactions. (3) Alteration of DNA methylation affects gene expression, leading to altered expression of genes involved in cell growth regulation, (4) which in turn promotes the growth of preexisting (spontaneously) initiated preneoplastic hepatocytes in B6C3F1 mouse liver and ultimately produces hepatic neoplasia (Fig. 11).
Several lines of experimental evidence support this proposed mode of action. Diethanolamine is known to disrupt intracellular choline homeostasis by inhibiting choline uptake (Lehman-McKeeman and Gamsky, 1999, 2000). Important to the proposed mechanism, inhibition of choline uptake and decreased phosphocholine synthesis were reversed by removing diethanolamine from cell culture medium and prevented by supplementing the culture medium with choline (Lehman-McKeeman and Gamsky, 1999, 2000). Furthermore, diethanolamine-induced morphological transformation in Syrian Hamster Embryo cells was prevented by choline supplementation (Lehman-McKeeman and Gamsky, 2000). In vivo, hepatic levels of choline metabolites and S-adenosyl methionine (SAM) were decreased in B6C3F1 mice dosed with diethanolamine, while no changes were observed in F344 rat liver (Lehman-McKeeman et al., 2002; Stott et al. 2000).
Choline depletion has been shown to function as a tumor promoter in rodents, and choline deficiency by itself is a nutritional deficiency considered carcinogenic in rodents (DeCarmargo et al., 1985; Newberne et al., 1982; Rogers et al., 1987). In vivo, choline that is not phosphorylated is oxidized to betaine, a product that serves as a methyl donor in a reaction that converts homocysteine to methionine. Methionine can then be converted to SAM, a methyl donor for many enzymatic reactions (Zeisel and Blusztajn, 1994). Since diethanolamine interferes with cellular choline uptake and results in choline depletion, diethanolamine treatment would be expected to reduce SAM levels and lower the potential for DNA methylation. Hypomethylation of DNA is considered an epigenetic mechanism of carcinogenesis and has been shown to activate a number of genes including oncogenes (Eden et al., 2003; Goodman and Watson, 2002). DNA hypomethylation was observed in rat liver after 7 days on a choline- or lipotrope-deficient diet (Locher et al., 1986; Wainfen et al., 1989). Thus, hypomethylation of critical target genes as a result of reduced availability of SAM could be a critical factor in the carcinogenic response observed in mice following diethanolamine exposure. Currently, studies are ongoing to determine the effect of diethanolamine on methylation status in mouse and rat hepatocytes.
The induction of DNA synthesis is an obligate event for the development of neoplasia. Choline deficiency is known to induce liver cell proliferation (Abanobi et al., 1982; Counts et al., 1996; Zeisel, 1996). In the current studies, DNA synthesis was increased 1.8- to 3.2-fold over control in mouse hepatocytes following exposure to diethanolamine at and above 10 μg/ml. Incubation of mouse hepatocytes in medium containing reduced choline concentrations (1/10 to 1/100 of normal medium; 0.898 mg/l to 0.0898 mg/l) for 24 h produced similar increases (1.4- to 2.4-fold) in DNA synthesis over control, whereas incubation of cells in medium containing 10-fold higher choline concentrations produced a 50% reduction in DNA synthesis compared to control groups. These results demonstrate that modulation of cellular choline levels modulates DNA synthesis in rodent hepatocytes.
In studies examining species selectivity of diethanolamine, increases in DNA synthesis were observed in mouse and rat, but not human hepatocytes following treatment with diethanolamine. Furthermore, incubation of hepatocytes in medium containing reduced choline increased DNA synthesis 1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively, while no increase was observed in human hepatocytes. Choline supplementation also reduced diethanolamine-induced DNA synthesis to control levels or below in mouse and rat hepatocytes. These results indicate that the induction of in vitro DNA synthesis by diethanolamine in the liver is associated with choline depletion, and is species specific. Similarly, diethanolamine elicited a dose-related increase in DNA synthesis in B6C3F1 mouse liver following dermal application of diethanolamine (160 mg/kg/d) for 1 and 13 weeks of exposure (Mellert et al., 2004). In mice dosed with diethanolamine for 1 week then allowed 3 weeks of recovery (no diethanolamine), hepatic labeling indices returned from 3-fold increase over control to control values, indicating that the proliferative effect of diethanolamine was reversible (Mellert et al., 2004). These findings provide further support that diethanolamine induces neoplasia through epigenetic mechanisms and functions at the promotion stage of the carcinogenesis process.
The present studies also examined the effects of diethanolamine and choline depletion on expression of genes involved in cell growth regulation in mouse and rat hepatocytes using cDNA microarrays. mRNA from mouse and rat hepatocytes treated with diethanolamine or choline depletion showed similar patterns of gene expression, altering several genes involved in pathways associated with cell cycle progression or suppression of apoptosis. In particular, p53 expression was decreased. This gene functions in several pathways including p21cip, which acts to inhibit cyclin D expression. Cyclin D acts on the Rb gene, in concert with E2F transcription factor, to drive the cell cycle. In the present experiments, both p53 and p21cip expression were decreased, while expression of Cyclin D1, Cyclin E, and E2F were increased. Therefore, the reduced p21cip block, coupled with increased cyclin and E2F expression, is permissive for entry into the cell cycle. Expression of p19, a gene encoding a protein that functions to inhibit cyclin D expression, was also decreased. In addition, caspase 3, bad, and bax expression exhibited a reduced expression. Reduction in expression of these pro-apoptotic genes would also be expected to reduce the number of cells undergoing apoptosis. Overall, the gene expression profile observed following treatment with diethanolamine or choline depletion treatment demonstrated an alteration of genes involved in pathways leading to a reduction in apoptosis as well as a progression into the cell cycle.
Chronic dermal exposure to diethanolamine resulted in a significant increase in liver neoplasia in the liver of treated mice but not the rat (National Toxicology Program, 1999). To date, no oral carcinogenicity studies with diethanolamine have been performed in rodents. In the present studies, diethanolamine altered expression of genes involved in cell growth regulation and induced DNA synthesis in rat hepatocytes. Following dermal application of diethanolamine in vivo, it has been shown that absorption of diethanolamine is greater in mice (25–60%) than in rats (3–16%) and results in approximately 3-fold higher blood and tissue concentrations of diethanolamine in mice compared to rats (Mathews et al., 1995, 1997; Mendrala et al., 2001). In the in vitro system used in the current studies, equimolar concentrations of diethanolamine were delivered to mouse and rat hepatocytes, whereas in vivo, due to differences in dermal absorption between species and differences in sensitivities to diethanolamine-induced skin irritation, blood and tissue concentrations of diethanolamine are lower in rats than in the mouse (Matthews et al., 1997). Thus, the concentrations of diethanolamine administered to rat hepatocytes were relatively high and were able to disrupt choline status and subsequently induce gene expression changes and DNA synthesis.
With respect to human risk from exposure to diethanolamine, the proposed mode of action for diethanolamine-induced choline deficiency is qualitatively applicable to humans. However, marked species differences in susceptibility to choline deficiency exist, with mice and rats being more susceptible than other species including humans, which are generally considered refractory to choline deficiency due to low levels of choline oxidase activity (Hoffbauer and Zaki, 1965; Sidransky and Farber, 1960; Zeisel and Blusztajn, 1994). Furthermore, diethanolamine absorption is considerably lower in humans compared to rodents. The differences in the absorptive properties of diethanolamine between species also correlates with the susceptibility to choline deficiency, with mice and rats being more susceptible than humans (Sun et al., 1996).
Several other hypotheses have been proposed for diethanolamine-induced hepatic neoplasia. Diethanolamine is a secondary amine that can undergo nitrosation to form a mutagenic nitrosamine, N-nitrosodiethanolamine. While N-nitrosodiethanolamine induced liver tumors in rats at doses of 2 mg/kg per day, this nitrosamine is not produced in measurable amounts following administration of diethanolamine at carcinogenic doses, suggesting that diethanolamine carcinogenicity does not result from N-nitrosodiethanolamine production (ECETOC, 1990; Preussmann, et al., 1981; Stott et al., 2000; Yamamoto et al., 1995). Due to structural similarity to choline and ethanolamine, diethanolamine also disrupts phospholipid metabolism, membrane function, and synthesis of fatty acid second messengers. Alterations in phospholipid metabolism in rats treated with diethanolamine, as well as the incorporation of diethanolamine into phospholipids and subsequent alteration of phospholipids have been reported (Barbee and Hartung, 1979; Mathews et al., 1995). However, the causality of these events to diethanolamine carcinogenicity has not been demonstrated.
Other effects associated with choline deficiency include increased generation of free radicals and increased susceptibility to oxidative damage (Floyd et al., 2002; Hensley et al., 2000; Rushmore et al., 1984) which may induce DNA damage and/or alter gene expression. An evaluation of reactive oxygen species or other oxidative stress parameters by diethanolamine has not been performed, and whether this mechanism participates in the induction of hepatic neoplasia by diethanolamine requires additional study.
Overall, the present results and those of other published studies support that the mode of action for diethanolamine hepatocarcinogenicity in mouse liver involves cellular choline deficiency that is associated with hypomethylation of DNA, alteration of gene expression, and increased DNA synthesis. Coupled together, these events result in the promotion of initiated cell populations in mouse liver. Further, the lack of induction of DNA synthesis by diethanolamine in human hepatocytes suggests that, due to species differences, humans are refractory to the carcinogenic effects of diethanolamine.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
This work was supported in part, by the Alkanolamines Panel of the American Chemistry Council (LMK), and by NIH R01CA100908 (J.E.K.). Conflict of interest: none declared.
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