ToxSci Advance Access originally published online on May 4, 2006
Toxicological Sciences 2006 92(2):378-386; doi:10.1093/toxsci/kfl007
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Mechanisms of 2-Butoxyethanol–Induced Hemangiosarcomas
Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
1 To whom correspondence should be addressed at Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS547, Indianapolis, IN 46202. Fax: (317) 274-7787. E-mail: jklauni{at}iupui.edu.
Received December 16, 2005; accepted April 19, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chronic exposure to 2-butoxyethanol increased liver hemangiosarcomas in male mice. The mechanism for the selective induction of hemangiosarcomas by 2-butoxyethanol is unknown but has been suggested to occur through non–DNA-reactive mechanisms. The occurrence of liver hemangiosarcomas in male mice has been linked to oxidative damage subsequent to RBC hemolysis and iron deposition and activation of macrophages (Kupffer cells) in the liver, events that exhibit a threshold in both animals and humans. 2-Butoxyethanol is metabolized to 2-butoxyacetaldehyde and 2-butoxyacetic acid, and although the aldehyde metabolite is short lived, the potential exists for this metabolite to cause DNA damage. The present study examined whether 2-butoxyethanol and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid, damaged mouse endothelial cell DNA using the comet assay. No increase in DNA damage was observed following 2-butoxyethanol (1–10mM), 2-butoxyacetaldehyde (0.1–1.0mM), or 2-butoxyacetic acid (1–10mM) in endothelial cells after 2, 4, or 24 h of exposure. Additional studies examined the involvement of hemolysis and macrophage activation in 2-butoxyethanol carcinogenesis. DNA damage was produced by hemolyzed RBCs (10 x 106, 4 h), ferrous sulfate (0.1–1.0µM; 2–24 h), and hydrogen peroxide (50–100µM; 1–4 h) in endothelial cells. Hemolyzed RBCs also activated macrophages, as evidenced by increased tumor necrosis factor (TNF)
, while neither 2-butoxyethanol nor butoxyacetic acid increased TNF- from macrophages. The effect of activated macrophages on endothelial cell DNA damage and DNA synthesis was also studied. Coculture of endothelial cells with activated macrophages increased endothelial cell DNA damage after 4 or 24 h and increased endothelial cell DNA synthesis after 24 h. These data demonstrate that 2-butoxyethanol and related metabolites do not directly cause DNA damage. Supportive evidence also demonstrated that damaged RBCs, iron, and/or products from macrophage activation (possibly reactive oxygen species) produce DNA damage in endothelial cells and that activated macrophages stimulate endothelial cell proliferation. These events coupled together provide the events necessary for the induction of hemangiosarcomas by 2-butoxyethanol. Key Words: 2-butoxyethanol; liver hemangiosarcomas; macrophages; hemolysis; DNA damage; endothelial cells.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-Butoxyethanol (ethylene glycol monobutyl ether), a widely used solvent in industrial and consumer products, has been shown to increase liver hemangiosarcomas in male B6C3F1 mice following chronic inhalation exposure (NTP, 2000
). No increase in hemangiosarcomas was observed in similarly exposed female mice or male and female rats. 2-Butoxyethanol was shown to be negative for DNA reactivity in standard genotoxicity assays and bacterial mutagenesis assays (Elliott and Ashby, 1997; Hoflack et al., 1995), suggesting that the liver tumor response results from indirect or nongenotoxic mechanisms. Metabolism of 2-butoxyethanol occurs through alcohol and aldehyde dehydrogenases, yielding 2-butoxyacetaldehyde and 2-butoxyacetic acid, respectively (Ghanayem et al., 1987a). Although 2-butoxyethanol itself was negative for genotoxicity (Elliott and Ashby, 1997; Hoflack et al., 1995), the question remains whether the aldehyde metabolite is capable of inducing DNA damage in the target tissue of 2-butoxyethanol carcinogenicity.
Oxidative damage and DNA synthesis have previously been proposed to be involved in the selective induction of neoplasia in the mouse liver. Increases in the oxidative DNA lesion 8-hydroxydeoxyguanosine (OH8dG) was detected in liver of mice subchronically exposed to 2-butoxyethanol; an increase that correlated with increased DNA synthesis (Siesky et al., 2002). In mouse hepatocytes, no increase in oxidative DNA damage was seen following treatment with 2-butoxyethanol or 2-butoxyacetic acid (Park et al., 2002b), suggesting that the induction of oxidative damage seen in vivo occurred through an indirect effect of 2-butoxyethanol exposure. Exposure to 2-butoxyethanol results in hemolysis of RBCs in rodents (Ghanayem et al., 1987b). To address an effect of hemolysis, iron was evaluated for its ability to induce oxidative damage in mouse hepatocytes and significantly increased OH8dG levels (Park et al., 2002b). In the Syrian hamster embryo (SHE) cell transformation assay, 2-butoxyethanol or 2-butoxyacetic acid failed to induce morphological transformation, whereas ferrous sulfate increased cellular transformation (Park et al., 2002b). Iron also increased DNA strand breaks in SHE cells, measured using the comet assay (Park et al., 2002b). These results provide evidence supporting the fact that the induction of oxidative DNA damage and carcinogenicity seen following 2-butoxyethanol exposure in vivo occurs through indirect or secondary effects rather than a direct effect of 2-butoxyethanol or metabolites.
A finding associated with 2-butoxyethanol–induced RBC hemolysis was iron deposition (hemosiderin) in Kupffer cells following chronic 2-butoxyethanol exposure in rodents (NTP, 2000). Kupffer cells, the resident macrophages of the liver, phagocytize foreign material (including damaged erythrocytes) and upon activation secrete biologically active products such as reactive oxygen species and cytokines (Knolle et al., 1995; Terpstra and van Berkel, 2000). Many products released from activated Kupffer cells (e.g., tumor necrosis factor [TNF] , IL-6, and reactive oxygen species) may participate in the carcinogenesis process through inducing oxidative stress and damage, altering signaling cascades, and/or increasing cell proliferation (Arii and Imamura, 2000; Bacon and Britton, 1990; Klaunig and Kamendulis, 2004). Iron is included among the list of compounds shown to activate Kupffer cells. In vitro, iron stimulated the release of TNF- and activation of NF
B from Kupffer cells (She et al., 2002).
The effects of 2-butoxyethanol observed in vivo and in vitro have lead to the hypothesis that 2-butoxyethanol–induced liver hemangiosarcomas in male mice results from Kupffer cell activation, secondary to RBC hemolysis (Fig. 1). Iron deposition following hemolysis and/or phagocytosis of damaged RBCs may result in activation of Kupffer cells, in turn resulting in the production and release of reactive oxygen species and cytokines. These factors may then produce oxidative DNA damage and induce cell proliferation in neighboring cell populations, such as the hepatic endothelial cells. The induction of cancer involves both mutational and cell proliferation events; as such, the present studies were designed to examine the role of the Kupffer cell in modulating 2-butoxyethanol–induced neoplasia by studying the effects of 2-butoxyethanol and related metabolites (1) on the induction of DNA damage in endothelial cells using the comet assay and (2) on macrophage activation. The effect of activated macrophages on endothelial cell DNA synthesis was also evaluated.
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MATERIALS AND METHODS |
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Chemicals.
2-Butoxyacetic acid was purchased from Spectrum Chemicals (Gardena, CA) and 2-butoxyacetaldehyde was obtained from R. Boatman (Eastman Kodak Co, Rochester, NY). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc (Logan, UT). 2-Butoxyethanol, hydrogen peroxide, ferrous sulfate, lipopolysaccharide (LPS), DMEM, and all other chemicals were purchased from Sigma Chemical Co (St Louis, MO).
Assessment of cytotoxicity.
Cytotoxicity was examined using the MTT (3-(4,5-dimethylthiazol-2-lyl)-2,5-diphenyl tetrazolioum) assay (Mosmann, 1983). MTT was added to endothelial cell cultures for the last 3 h of treatment (final concentration of 0.5 mg/ml). Media was then removed and the dye dissolved with 2-isopropanol (30 min, 37°C). Supernatants (100 µl) were transferred to a 96-well plate, and the optical density was measured at 570 nm with a reference at 630 nm. The highest noncytolethal concentrations (2-butoxyethanol, 10mM; 2-butoxyacetaldehyde, 1mM; and 2-butoxyacetic acid, 10mM) were used as the highest concentration evaluated in subsequent experiments. Although it is difficult to compare concentrations used in vitro to blood and tissue levels achieved following in vivo dosing, the concentrations used in the present studies were higher than those observed following in vivo exposure (Deisinger and Boatman, 2004; Dill et al., 1998; Lee et al., 1998).
Cell culture.
SVEC4-10 mouse endothelial and RAW 264.7 mouse macrophage lines were purchased from ATCC (Manassas, VA). The SVEC4-10 endothelial cell line was derived from the mouse and maintain properties associated with primary endothelial cells, including expression of VCAM and factor VIII, and express MHC class II antigen following IFN-
stimulation similar to that of normal endothelial cells. The RAW 264.7 macrophage line was derived from the mouse and is similar to primary mouse macrophages in that they are able to phagocytize latex beads, are activated by LPS, and express TNF-. SVEC4-10 cells were cultured in DMEM supplemented with 10% FBS, penicillin (200 U/ml), streptomycin (200 µg/ml), and amphotericin B (25 µg/ml), at 37°C in 5% CO2 and 95% relative humidity. Cells were plated (1.75 x 105 per 35 mm2 dish) and allowed to attach for 24 h prior to chemical treatments.
RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, penicillin (200 U/ml), streptomycin (200 µg/ml), and amphotericin B (25 µg/ml), at 37°C in 5% CO2 and 95% relative humidity. Macrophages were plated (1 x 106 per 35 mm2 well) and allowed to attach for 24 h. Cells were then washed (HBSS, 2x) and cultured in DMEM supplemented with 1% FBS and antibiotics at 37°C in 5% CO2 and 95% humidity.
Coculture experiments.
SVEC4-10 cells were cultured as above, plated (1.75 x 105 per 35 mm2 dish), and allowed to attach for 24 h. RAW 264.7 cells were cultured as described above, plated at 1 x 105 per cell culture insert (3.0 µm pore size), and allowed to attach for 24 h. After 24 h, cells were washed (HBSS, 2x) and cultured in DMEM supplemented with 1% FBS and antibiotics at 37°C in 5% CO2 and 95% humidity. Macrophages were treated with LPS or 10 x 106 hemolyzed mouse RBCs for 4 h and then washed (HBSS, 2x). This concentration of hemolyzed RBCs was previously shown to induce oxidative damage in hepatocytes (Park et al., 2002a). Inserts were transferred to endothelial cell cultures and incubated in 1 ml culture medium for up to 24 h for the assessment of DNA damage (comet assay) or DNA synthesis.
Comet assay: single-cell gel electrophoresis.
The comet assay was performed as described (Rojas et al., 1999; Singh et al., 1988) with modifications. Briefly, cells were washed (PBS, 2x), scraped, and centrifuged (600 x g, 5 min) and resuspended in PBS (1 ml, 1x). Cell suspensions were added to 1% low-melting agarose (42°C) and applied to Trevigen CometSlides (Gaithersburg, MD). After 40 min, slides were lysed (100mM Na2EDTA, pH 10, 2.5M NaCl, 10mM Trizma-base, 1% sodium lauryl sarcosinate, 1% Triton-X 100; 1 h, 4°C), placed in alkali buffer (0.3M NaOH, 1mM Na2EDTA, pH > 13; 40 min, 4°C), and then electrophoresed in alkali buffer (25 V, 300 mA, 30 min). Slides were then neutralized (0.4M Trizma-HCl, pH 7.5; 5 min), rinsed in deionized water (10 min), and allowed to dry overnight. Prior to visualization, slides were stained with ethidium bromide (25 µl; 20 µg/µl) and covered with a cover slip. A total of 100 nuclei per treatment was selected at random and measured with a Nikon fluorescence microscope and Komet 4.0 imaging Software (Kinetic Imaging Ltd, South Windsor, CT). DNA damage was expressed as comet (Olive) tail moment [(tail mean – head mean) tail %DNA/100] (Rojas et al., 1999; Tice et al., 1991).
Quantitation of DNA synthesis.
DNA synthesis was measured as described, with modifications (James and Roberts, 1996). BrdU (20µM) was added to cell cultures for the last hour of treatment. Medium was removed, and cells were washed, methanol fixed (70%, 10 min), and then processed for immunohistochemical staining of BrdU. Incorporated BrdU was localized using an anti-BrdU antibody followed by a peroxidase-linked secondary antibody and a DAB substrate and then counterstained with hematoxylin. Replicative DNA synthesis (labeling index) was calculated by scoring the percentage of BrdU-positive endothelial cells (red-pigmented nuclei) in a minimum of 1000 cells.
TNF- determination.
Release of TNF- into culture medium was measured in RAW cells following exposure to chemicals for 4 h. TNF- was quantified spectrophotometrically (450 nm) using a Biosource mouse TNF- CytoSet ELISA kit (BioSource, Carlsbad, CA). Sample concentrations were determined from a standard curve generated using a four-parameter curve fit algorithm.
Statistics.
For studies examining DNA damage, DNA synthesis, and TNF-, differences between groups were evaluated using ANOVA and the Student t test with a level of significance at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of 2-Butoxyethanol, 2-Butoxyacetaldehyde, and 2-Butoxyacetic Acid on Endothelial Cell DNA Damage Using the Alkaline Comet Assay
To examine whether 2-butoxyethanol, 2-butoxyacetaldehyde, or 2-butoxyacetic acid induced DNA strand breaks, the single-cell gel electrophoresis (comet) assay was performed in mouse SVEC4-10 endothelial cells. Cells were treated with 2-butoxyethanol (1, 5, or 10mM), 2-butoxyacetaldehyde (0.1, 0.5, or 1mM), or 2-butoxyacetic acid (1, 5, or 10mM) for 2, 4, or 24 h. These concentrations are higher than expected following an in vivo exposure. Following a 600-mg/kg dose of 2-butoxyethanol via gavage, the peak concentration of 2-butoxyacetacldehyde achieved in blood or tissues was 33µM (Deisinger and Boatman, 2004
). Similarly, the concentrations of 2-butoxyethanol and 2-butoxyacetic acid observed in mice following inhalation of 125 ppm 2-butoxyethanol resulted in low (µM) levels of 2-butoxyethanol and 2-butoxyacetic acid, much lower than the concentrations (mM) used in the present studies (Dill et al., 1998; Lee et al., 1998). 2-Butoxyethanol, 2-butoxyacetaldehyde, or 2-butoxyacetic acid did not produce significant increases in DNA damage (comet tail moment) relative to control at any time point examined in endothelial cells (Table 1). Two agents were used as positive controls, hydrogen peroxide and benzo[a]pyrene. Hydrogen peroxide was selected as a positive control at the 2- and 4-h treatments because of its production during macrophage activation. Benzo[a]pyrene was used for the 24-h treatments as a positive control because of its genotoxicity and due to the fact that hydrogen peroxide effects dissipate by 6 h in our model. As shown (Table 1), hydrogen peroxide consistently increased DNA damage in endothelial cells at these time points (
6- to 10-fold at 2 h and 3-fold at 4 h) while benzo[a]pyrene produced significant increases in DNA damage in all experiments ( 1.6- to 2.0-fold; Table 1).
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Effect of Hemolyzed RBCs, Ferrous Sulfate, and Hydrogen Peroxide on Endothelial Cell DNA Damage Using the Alkaline Comet Assay
DNA strand breaks were measured in mouse endothelial cells treated with hemolyzed mouse RBCs (0.1 x 106, 1.0 x 106, or 10 x 106) for 2, 4, and 24 h (Table 2); ferrous sulfate (0.1, 0.5, or 1µM) for 2, 4, or 24 h (Table 2); or hydrogen peroxide (10–100µM) for 1, 2, or 4 h (Fig. 2). In contrast to 2-butoxyethanol and metabolites, hemolyzed RBCs (10 x 106) increased DNA damage after 4 h (24% increase in comet tail moment; Table 2). No significant increase in DNA damage was seen at other concentrations or after 2 or 24 h (Table 2). Ferrous sulfate produced concentration-related increases in comet tail moment (Table 2). Significant increases in endothelial cell DNA damage was observed at concentrations of 0.5 and 1.0µM ferrous sulfate after 2 and 4 h. All concentrations of ferrous sulfate examined (0.1–1.0 µM) increased DNA damage in endothelial cells after 24 h (1.7- to 2.2-fold; Table 2). Increases in comet tail moment were also evident following treatment with 50–100µM hydrogen peroxide after 1 h (approximately five- to eightfold) and 75 and 100µM after 2 and 4 h (Fig. 2). DNA damage by hydrogen peroxide decreased over time (Fig. 2) with no increase in DNA damage observable following treatment for 24 h (data not shown).
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Effect of 2-Butoxyethanol, 2-Butoxyacetic Acid, Hemolyzed RBCs, and Ferrous Sulfate on Macrophage Activation
To determine whether 2-butoxyethanol, 2-butoxyacetic acid, hemolyzed RBCs, or ferrous sulfate activated mouse macrophages, release of TNF-
into cell culture medium was measured using ELISA. Mouse RAW 264.7 macrophages were treated with 2-butoxyethanol or 2-butoxyacetic acid (1, 10, or 25mM), hemolyzed RBCs (0.1 x 106, 1.0 x 106, or 10 x 106), or ferrous sulfate (50, 250, or 500µM) for 4 h. Higher concentrations of ferrous sulfate were used in this experiment compared with those used in endothelial cell studies as ferrous sulfate was without effect on cytolethality at these concentrations and for this treatment duration in macrophages. No increase in TNF- was observed following treatment with 2-butoxyethanol, 2-butoxyacetic acid, or ferrous sulfate after 4 h of exposure (Fig. 3). Hemolyzed RBCs (10 x 106), however, resulted in a significant increase in TNF- release from macrophages (approximately fivefold; Fig. 3). LPS was used as a positive control in all studies and produced significant increases of TNF- in culture medium after a 4-h exposure ( 60- to 90-fold increase; data not shown).
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Effect of Activated Macrophages on Endothelial Cell DNA Damage Using the Alkaline Comet Assay
Studies were performed to determine whether the products released from activated macrophages were capable of producing DNA damage in endothelial cells. In these studies, macrophages were treated with either LPS (10 ng/ml) or hemolyzed RBCs (1 x 106) for 4 h. Following activation, remaining treatments were removed from macrophage cultures by washing with HBSS. Activated macrophages were then placed in coculture with endothelial cells for 4 or 24 h, and the comet assay was then performed on endothelial cells. Significant increases in DNA damage was seen in endothelial cells cocultured with activated macrophages after 4 h (1.6-fold increase) and after 24 h (twofold increase; Fig. 4). DNA damage was not significantly altered in endothelial cells cocultured with nonactivated macrophages after 4 or 24 h (Fig. 4). Macrophages activated with hemolyzed RBCs (10 x 106) for 4 h did not increase endothelial cell DNA damage after 4 or 24 h (data not shown).
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Effect of Activated Macrophages on Endothelial Cell DNA Synthesis
The ability of the products released from activated macrophages to induce DNA synthesis in endothelial cells was also evaluated. Macrophages were treated with either LPS (10 ng/ml) or hemolyzed RBCs (10 x 106) for 4 h. Following activation, remaining LPS or hemolyzed blood was removed from macrophage cultures by washing with HBSS. Activated macrophages were then placed in coculture with endothelial cells for 24 h. Coculture of endothelial cells with activated macrophages significantly increased endothelial cell DNA synthesis (34–48% increase with hemolyzed RBCs and LPS, respectively; Table 3). Basic fibroblast growth factor (bFGF) served as a positive control and resulted in a significant induction of DNA synthesis (31% increase; Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-Butoxyethanol produced hepatic hemangiosarcomas following chronic exposure in male B6C3F1 mice (NTP, 2000
). Additionally, 2-butoxyethanol resulted in hemolysis in mice and increased hemosiderin in Kupffer cells (Ghanayem et al., 1987a; NTP, 2000). These observations coupled with negative mutagenicity and genotoxicity data (Elliott and Ashby, 1997; Hoflack et al., 1995) lead to the development of a mode of action for 2-butoxyethanol–induced hemangiosarcomas that is centered on hemolysis of RBCs via 2-butoxyacetic acid and activation of Kupffer cells, events that ultimately lead to endothelial cell proliferation and hemangiosarcomas (Klaunig and Kamendulis, 2005; Park et al., 2002b; Siesky et al., 2002, Fig. 1). The studies presented in this article provide further evidence supporting this proposed mode of action.
Metabolism of 2-butoxyethanol occurs through alcohol and aldehyde dehydrogenases to yield 2-butoxyacetaldehyde and 2-butoxyacetic acid, respectively (Ghanayem et al., 1987b). Aldehyde metabolites are typically considered reactive compounds, and as such, the possibility that the aldehyde metabolite could induce damage to DNA and/or mutation was questioned. DNA damage, assessed using the comet assay, in endothelial cells treated with 2-butoxyethanol and the major metabolites 2-butoxyacetaldehyde and 2-butoxyacetic acid revealed that none of these compounds were directly able to produce endothelial cell DNA damage. Assessment of the pharmacokinetic data for 2-butoxyethanol metabolism supports that 2-butoxyacetaldehyde is a transient, labile intermediate metabolite and thus may not achieve concentrations sufficient to cause DNA damage in target tissues (Deisinger and Boatman, 2004). These results support the fact that due to a lack of DNA reactivity, 2-butoxyethanol and related metabolites are not likely to produce new mutations in endothelial cells and agree with previous results demonstrating that 2-butoxyethanol is devoid of direct mutagenicity and genotoxicity (Elliott and Ashby, 1997; Hoflack et al., 1995).
Hemolysis of RBCs is considered the primary nonneoplastic response observed following 2-butoxyethanol exposure (NTP, 2000). Iron is a prooxidant capable of producing oxidative stress through catalyzing the conversion of the superoxide anion and hydrogen peroxide to the highly reactive hydroxyl radical via the Fenton reaction (Aust et al., 1985; Halliwell and Gutteridge, 1984). When iron storage capacity is exceeded in vivo, the metal can accumulate in tissues such as parenchymal cells in the liver, resulting in iron overload (Ley et al., 1982; Searle et al., 1987). Iron overload in humans, whether genetic or by dietary intake, promotes free radical–induced oxidative damage which increases the potential for functional alterations of biomolecules and organelles (Aust et al., 1985; Britton et al., 1987) and has been associated with the induction of hepatic cancer (Mandishona et al., 1998; Niederau et al., 1985; Stevens et al., 1994). The present studies demonstrated that both iron (ferrous sulfate) and hemolyzed RBCs, although the latter to a much lower extent, increased endothelial cell DNA damage (Table 2). These results demonstrate that the products of hemolyzed blood are capable of inducing DNA damage in endothelial cells and suggest that the hemolysis produced following 2-butoxyethanol exposure may result in DNA damage and the induction of mutations in endothelial cells.
A second key step in the proposed mode of action for 2-butoxyethaol–induced hemangiosarcomas is the activation of Kupffer cells following 2-butoxyethanol exposure and hemolysis of RBCs. The observations that 2-butoxyethanol causes hemolysis via 2-butoxyacetic acid (Bartnik et al., 1987; Ghanayem, 1989; Ghanayem and Sullivan, 1993; Ghanayem et al., 1987b) and results in an accumulation of hemosiderin (iron deposition) in the mouse liver (Krasavage, 1986) are supportive of this step, as these events may act as stimuli capable of activating Kupffer cells. Of qualitative importance to human risk, many studies have documented that humans are significantly less sensitive to the hemolytic effects of 2-butoxyacetic acid than rats or mice (Ghanayem and Sullivan, 1993; Udden, 1994, 2002; Udden and Patton, 1994).
An association between Kupffer cell pigmentation (hemosiderin) caused by hemolysis and liver hemangiosarcomas has been reported in male mice for a limited group of chemicals including 2-butoxyethanol (NTP, 2000; Nyska et al., 2004). Nyska et al. (2004) further suggested that RBC hemolysis results in the formation of reactive oxygen species and that the sex-specific increase in hemangiosarcomas seen with these chemicals was likely related to hormonal differences or reduced antioxidant defense capacity in male mice (Nyska et al., 2004). In addition to sex-related differences in the mouse for the induction of hemangiosarcomas, 2-butoxyethanol appears to be species selective and does not result in hemangiosarcomas in the rat (NTP, 2000). Previously, we demonstrated a selective induction of oxidative stress by 2-butoxyethanol; oxidative damage was observed in male mouse liver but not in rat liver (Siesky et al., 2002). The selective induction of oxidative DNA damage may be related to lower hepatic levels of the antioxidant vitamin E in the mouse liver compared to the rat (Siesky et al., 2002; Park et al., 2002a). Furthermore, a canvas of the NTP bioassay database has revealed that the mouse has a relatively high background of spontaneous endothelial neoplasms in the liver compared to the rat, a species in which no liver hemangiosarcomas have been reported in control animals (Klaunig and Kamendulis, 2005).
The present studies demonstrated that 2-butoxyethanol and 2-butoxyacetic acid did not directly activate macrophages, as evidenced by a lack of TNF- production from macrophages. Iron (ferrous sulfate) also failed to increase TNF- release from macrophages. This result is in contradiction to a previous report demonstrating that iron increased TNF- levels in primary isolated Kupffer cells (She et al., 2002). Although both cell types are macrophages and would be expected to respond to activating stimuli similarly, these results may be attributable to differences between the macrophage line and primary isolated Kupffer cells. Hemolyzed RBCs were shown to activate macrophages, producing a marked induction of TNF- in macrophages. These results support the proposed mode of action and suggest that macrophage activation does not result from free iron, but rather that heme and/or hemolyzed RBCs stimulate macrophage activation following 2-butoxyethanol–induced hemolysis.
Activated Kupffer cells are a source of both proinflammatory cytokines and reactive oxygen and nitrogen species that elicit a number of biological and pathological effects (Arii and Imamura, 2000; Decker, 1990). Excess production of reactive oxygen species, whether derived from activated macrophages or through Fenton chemistry, can induce a variety of DNA damage including oxidative modifications and strand breaks (Dizdaroglu, 1992). The hydroxyl radical, while highly reactive, has an extremely short half-life, whereas hydrogen peroxide and superoxide anion, although less reactive, are more biologically persistent. However, only hydrogen peroxide can easily diffuse across cell membranes (Parola and Robino, 2001) and thus may be considered a representative oxidant to study the effects of reactive oxygen species on endothelial cell DNA damage. The current studies showed concentration-related increases in DNA damage by hydrogen peroxide in endothelial cells. Interestingly, the damage appeared to decrease over time, suggesting that DNA repair may have been stimulated to compensate the oxidative burden in the treated endothelial cells. These results are in agreement with those of Jaruga and Dizdaroglu (1996) who noted that the majority of hydrogen peroxide–induced DNA damage was repaired within 6 h in mammalian lymphoblast cells. Overall, the present results support the fact that products of hemolyzed blood and reactive oxygen species are capable of inducing DNA damage in endothelial cells and may provide a mechanism for induction of new mutations in this cell type.
As it was previously demonstrated that reactive oxygen species can induce DNA damage, experiments were performed in which macrophages were activated with either LPS or hemolyzed RBCs and then placed in coculture with endothelial cells. This experimental design allowed for the effects of products released from the activated macrophage to be directly assessed in target endothelial cell populations without the activating agent confounding the interpretation of results. LPS-activated macrophages increased in endothelial cell DNA damage, further supporting a role for macrophage activation in the induction of DNA damage in adjacent cell populations, such as endothelial cells. However, hemolyzed RBC–activated macrophages did not induce endothelial cell DNA damage. Although this result may appear to be in opposition of the proposed mode of action, the lack of effect may have been due to the relatively low level of activation produced by hemolyzed RBCs (Fig. 3) and/or the exposure duration of macrophages to hemolysis (4 h). Since hemolysis occurs throughout the entire treatment during 2-butoxyethanol exposure in vivo, the 4-h exposure of macrophages to hemolyzed RBCs may not be representative of the events and biological responses that are elicited by 2-butoxyethanol in vivo.
While excess production of reactive oxygen species produces DNA damage, an important biological response to low levels of reactive oxygen species is the stimulation of cell-signaling pathways and modulation of cell proliferation, in particular at the tumor promotion stage of carcinogenesis (Cerutti, 1985; Klaunig and Kamendulis, 2004). In addition to reactive oxygen species, activated macrophages produce a number of growth-stimulatory cytokines, such as IL-6 and IL-1, as well as vascular endothelial growth factor (VEGF) (Arii and Imamura, 2000; Decker, 1990; Sunderkotter et al., 1994). As such, the products released following macrophage activation may be associated with neoplastic development. In particular, macrophage-derived VEGF may be an important regulatory molecule for the induction of liver hemangiosarcomas and warrants further investigation.
Cell proliferation is involved in the carcinogenesis process by allowing for the clonal growth of mutated cells and/or preneoplastic cell populations and may also incorporate new mutations in cells undergoing DNA replication. Therefore, measurement of cell proliferation is important in the study of carcinogenesis. Because the synthesis phase of the cell cycle replicates nuclear DNA, its frequency is regarded as an indicator of the cell proliferation response (Goldsworthy et al., 1993). The present studies provided an additional linkage between the products of activated macrophages and the induction of DNA synthesis in endothelial cells. Macrophages activated by either LPS or hemolyzed RBCs that were then cocultured with endothelial cells resulted in an increase in endothelial cell DNA synthesis.
These results demonstrate that 2-butoxyethanol and its major metabolites (2-butoxyacetaldehyde and 2-butoxyacetic acid) do not directly damage endothelial cell DNA, while products from activated macrophages induced endothelial cell DNA damage and DNA synthesis. These studies support the mode of action proposed for the induction of hemangiosarcomas following 2-butoxyethanol exposure (Klaunig and Kamendulis, 2005; Park et al., 2002b; Siesky et al., 2002, Fig. 1) and further suggest that the induction of neoplasia is secondary to RBC hemolysis, an event that exhibits a threshold in both animals and humans and arises in part due to the activation of hepatic macrophages.
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ACKNOWLEDGMENTS |
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This study was supported in part by grants from the NIH/NCI (R01-CA100908; J.E.K.) and the American Chemistry Council (J.E.K.).
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