Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS 1021, Indianapolis, Indiana 46202
Received June 4, 2002; accepted August 12, 2002
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ABSTRACT |
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Key Words: 2-butoxyethanol; 2-butoxyacetic acid; DNA synthesis; oxidative stress; hepatocytes; endothelial cells; liver; BrdU; cell proliferation; iron; Kupffer cells.
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INTRODUCTION |
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2-Butoxyethanol exposure resulted in hemolysis in rodents via 2-butoxyacetic acid, the major metabolite of 2-butoxyethanol (Ghanayem et al., 1987a, 1990
). Associated with 2-butoxyethanol-induced hemolysis in the rodents was an increase in hemosiderin (iron deposition) in Kupffer cells (NTP, 2000
), presumably arising from red blood cell hemolysis (Babior and Stossel, 1994
). Iron, via Fenton and Haber-Weiss reactions, can produce reactive oxygen species including hydroxyl radicals that in turn may induce lipid peroxidation or oxidative DNA damage and contribute to carcinogenesis (Bacon and Britton, 1990
). Excess uptake of iron has also been shown to increase the growth of cancer cells (Bergeron et al., 1985
) as well as increase the conversion of preneoplastic lesions to neoplasia by chemical carcinogens (Siegers et al., 1988
). A role for iron in the cancer process is also suggested by results showing a reduction in neoplasm induction by lowered iron concentration (Thompson et al., 1991
). Free iron has been shown to activate Kupffer cells, resulting in the release of reactive oxygen species and cytokines. These biologically active compounds may participate in the carcinogenesis process. In particular, tumor necrosis factor
, released from activated Kupffer cells, has been associated with the upregulation of cell proliferation (Arii and Imamura, 2000
).
Our working hypothesis of 2-butoxyethanol-induced liver neoplasms in male mice is founded on the induction of oxidative damage and Kupffer cell activation secondary to red blood cell hemolysis. The oxidative stress arises from iron deposition from 2-butoxyethanol-induced hemolysis and resulting iron deposition in the Kupffer cells. This in turn results in the production of reactive oxygen species and the release of cellular cytokines that produce oxidative DNA damage and induce cell proliferation in the two target cell types (endothelial cell and hepatocytes) of 2-butoxyethanol-induced liver carcinogenesis (Fig. 1). The induction of cancer involves both mutational and cell proliferation events. Therefore, this study investigated whether subchronic exposures to 2-butoxyethanol in rats and mice resulted in an increase in oxidative damage (DNA damage and lipid peroxidation) and DNA synthesis in endothelial cells and hepatocytes selectively in the mouse liver.
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MATERIALS AND METHODS |
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Animals.
B6C3F1 male mice (68 weeks of age) were purchased from Charles Rivers Laboratories (Wilmington, MA). F344 male rats (68 weeks of age) were purchased from Harlan (Indianapolis, IN). Animals were acclimated for 2 weeks prior to exposure to 2-butoxyethanol. Mice were housed five per cage and rats two per cage in polycarbonate cages with filter tops in the Indiana University AAALAC-certified animal facility. All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All animals received NIH-07 pelletized diets and de-ionized water ad libitum.
Experimental design.
Mice were randomly placed into four treatment groups (60 mice/group). Rats were randomly placed into three treatment groups (20 rats/group). Mice received 2-butoxyethanol via daily gavage (5 times per week) at 0, 225, 450, or 900 mg/kg body weight (b.w). Rats received 2-butoxyethanol via daily gavage (5 times per week) at 0, 225, or 450 mg/kg b.w. 2-Butoxyethanol was dissolved in deionized water. All treatment doses were prepared fresh weekly. A dose range-finding study was initially performed to determine the maximum tolerated dose (data not shown). Gavage doses used correlated with those of a previous inhalation study (NTP, 2000). After exposure for 7, 14, 28, or 90 days, 15 mice and 5 rats were sampled from each treatment group.
Seven days prior to sacrifice, an osmotic minipump was implanted in five animals from each group (mouse: Model 2001, rat: Model 2ML1, Alzet Co., Palo Alto, CA). Minipumps were filled with BrdU (20 mg/ml 0.9% sterile saline) for the determination of DNA synthesis. At sampling, animals were killed by asphyxiation, weighed, and necropsied. Livers were perfused with 1x PBS, removed, and weighed. A portion of liver from each animal was fixed in formalin, embedded in paraffin, and sectioned. The remaining liver from each animal was snap frozen in liquid nitrogen and stored at 80°C for analysis of oxidative stress end points.
Hematocrit.
Blood was collected by cardiac puncture at necropsy and transferred to microcapillary tubes. Samples were centrifuged (10 min, 7800 rpm, 930 hematocrit head rotor; International Equipment Co., Needham Heights, MA) and the percent hematocrit determined (percent red blood cells in total blood volume) using a circular microcapillary tube reader (No. 2201, IEC, Needham Heights, MA) for each animal (Wintrobe, 1981).
Perl (iron) staining and quantification.
Sections of liver were stained for ferric iron using the Perl (Prussian Blue) method (Bugelski, 1985). Positive Perl staining was localized to Kupffer cells. Kupffer cells were counted in each of the liver lobes for each animal (approximately 2000 cells). A Perl index was determined by dividing the total number of labeled Kupffer cells (Perl-positive stain) by the total number of cells counted x 100.
Determination of 8-hydroxydeoxyguanosine (OH8dG).
OH8dG was measured using isolated DNA from the livers of treated animals and control animals. DNA was isolated by sodium iodide (NaI) chaotropic extraction for the analysis of OH8dG (Wang et al., 1994). Briefly, tissue (
100 mg) was homogenized and centrifuged. The supernatant was then digested with proteinase K and RNase A. Following digestion, DNA was precipitated with NaI and ice-cold isopropyl alcohol and centrifuged. DNA then was dissolved in Tris-HCl, sequentially digested with nuclease P1 and alkaline phosphatase, and centrifuged. Two hundred µl of supernatant was used for HPLC analysis. Elution was carried out with a mobile phase consisting of 100mM sodium citrate using a Waters Nova-Pak C18 reversed-phase analytical column on a Waters 2690 Alliance HPLC System (Waters, Milford, MA). OH8dG was detected electrochemically (ESA Coularray, 12-channel; ESA, Chelmsford, MA) and 2'-deoxyguanosine (dGuo) was detected at 250 nm (WatersTM 996 system, Milford, MA). OH8dG and dGuo were quantitated from standards prepared in the mobile phase immediately before sample analysis. Results were expressed as µmol OH8dG/mol dGuo.
Malondialdehyde determination.
Malondialdehyde was measured in liver as previously described (Bagchi et al., 1993). Briefly, liver (
100mg) was homogenized with perchloric acid and centrifuged. The supernatant was then derivatized with 2,4-dinitrophenylhydrazine. The aqueous phase was extracted with equal volumes of pentane (2x), and the pentane was then evaporated under nitrogen. Residue was dissolved in 200 µl mobile phase (49% aqueous acetonitrile, pH 7.2) and eluted using a Waters Nova-Pak C18 reversed-phase analytical column (Waters 2690 Alliance HPLC System, Waters, Inc., Milford, MA). Malondialdehyde was detected at 330 nm (Waters 484 Tunable Absorbance Detector, Waters, Inc., Milford, MA). Malondialdehyde concentrations were calculated from a standard curve prepared in mobile phase immediately before sample analysis and expressed as nmol malondialdehyde/g liver.
Quantitation of DNA synthesis.
Immunohistochemical detection of BrdU in liver was performed as previously described (Eldridge et al., 1990). Cells that incorporated BrdU were visualized by the accumulation of red pigment in the nuclei compared with the counterstained blue nuclei (hematoxylin). Cells in 15 randomly selected fields were counted in each liver lobe for each animal (approximately 50006000 cells). The labeling index was determined in both endothelial cells and hepatocytes by dividing the total number of labeled cells by the total number of cells counted x 100. The identity of endothelial cells was confirmed by staining a serial section with anti-factor VIII as previously described (Sharifi et al., 2000
).
Apoptosis and mitosis.
Apoptosis and mitosis in the liver was defined and quantitated as previously described (Bursch et al., 1985). The identification of apoptotic cells was confirmed using TUNEL (Trevigen TAC XL Blue Label in Situ Apoptosis Detection Kit). Cells in 15 randomly selected fields were counted in each liver lobe for each animal (approximately 50006000 cells). Apoptotic and mitotic indices were determined by dividing the total number of labeled cells by the total number of cells counted x 100.
Statistical analysis.
Statistical differences in oxidative stress measurements (OH8dG, malondialdehyde, and vitamin E) were determined using the independent samples T-test (SPSS 10.0 software, Chicago, IL). Statistical analysis of all other measurements was performed using ANOVA followed by Dunnetts post hoc test (Gad and Weil, 1986). Values were considered statistically different from control at p < 0.05.
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RESULTS |
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DISCUSSION |
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2-Butoxyethanol, via the action of 2-butoxyacetic acid, induces hemolysis in rats and mice (Ghanayem and Sullivan, 1993). In this study, subchronic exposure to 2-butoxyethanol resulted in a decrease in hematocrit in both the mouse and rat at all time points examined. Furthermore, spleen weight was increased following treatment with 2-butoxyethanol in the mouse and rat, reflective of the accumulation of damaged red blood cells from the circulation (Ghanayem et al., 1992
). Chronic exposure to 2-butoxyethanol also increased hemosiderin pigmentation in Kupffer cells in mouse and rat liver (NTP, 2000
), apparently related to hemolysis. In this study, histological examination of liver sections revealed a dose-dependent increase in iron staining, localized to Kupffer cells, by 2-butoxyethanol in both mouse and rat liver. These findings support the proposal that the doses of 2-butoxyethanol used in this study resulted in hemolysis and iron deposition in Kupffer cells in both the rat and mouse.
Our working proposal is that the induction of neoplasia by 2-butoxyethanol occurs indirectly through the induction of oxidative stress and is driven by iron deposition in Kupffer cells from red blood cell hemolysis. Iron deposition in the liver, such as that seen with hemochromatosis, results in hepatic injury, causing fibrosis and cirrhosis in the liver and ultimately resulting in hepatocellular carcinoma (Bacon and Britton, 1990). The mechanism for the induction of cell injury by iron may be related to oxidative stress and damage. Iron catalyzes Fenton and Haber-Weiss reactions, resulting in the production of reactive oxygen species (Bacon and Britton, 1990
) that can result in oxidative damage. The production of oxidative stress and damage has been linked to the development and progression of cancer (Guyton and Kensler, 1993
). OH8dG, a predominant oxidized DNA base, has been used as an index of oxidative DNA damage (Kaneko et al., 1997
). Oxidative lipid damage (lipid peroxidation) may result in structural and functional changes to cellular membranes and ultimately lead to cell death or alteration of cell function or viability. In this study, a biphasic increase in OH8dG and lipid peroxidation was seen in the mouse.
Correlating with the increase in oxidative stress was a biphasic increase in DNA synthesis in the target cell types of 2-butoxyethanol-induced neoplasia (endothelial cell and hepatocytes). The endothelial cell DNA synthesis increase occurred soon after treatment (7 and 14 days), whereas the increase in hepatocyte DNA synthesis was seen only following 90 days of treatment. Many nongenotoxic hepatic carcinogens also induce cell proliferation (Butterworth, 1990; Goldsworthy et al., 1993
; Klaunig et al., 1998
). Increased cell replication may increase the frequency of spontaneous mutations through increasing the frequency of errors in DNA repair or replication. Alternatively, it may alter methylation of the genome and facilitate clonal expansion of initiated cells via a change in gene expression that either silences tumor suppressor genes or increases the expression of oncogenes, either of which may result in the formation of hepatic focal lesions (Watson and Goodman, 2002
). The observed increase in liver hemangiosarcoma following chronic inhalation to 2-butoxyethanol may be related to increased oxidative damage and DNA synthesis. The induction of DNA synthesis may promote already modified or mutated endothelial cells or produce an increase in endogenous mutations in the endothelial cells, resulting in the formation of new initiated cells. The target cell for neoplastic development may be differentiated liver cells. However, one cannot exclude a role for liver stem cells in this process (Knight et al., 2000
; Sell, 1993
; Sigal et al., 1992
). Although a statistical increase in the induction of hemangiosarcomas was seen in the chronic bioassay, the incidence of tumors was low (4/49 male mice). Therefore, the subtle increases observed in oxidative damage and DNA synthesis in the liver may relate to the mechanism for tumor induction by 2-butoxyethanol in the mouse. In contrast to the mouse liver, no increases in oxidative damage or DNA synthesis were observed in 2-butoxyethanol-treated rat liver. This finding further substantiates the involvement of oxidative damage and DNA synthesis in the selective induction of neoplasia in the mouse liver.
One possibility for the appearance of DNA synthesis and oxidative stress in mouse hepatocytes only after 90 days of exposure may involve changes that occur with respect to 2-butoxyethanol in the aged rodent. Older rats are more sensitive to the hemolytic effects of 2-butoxyethanol than younger rats (Carpenter et al., 1956; Ghanayem et al., 1987a
). In addition, older rodents exhibit compromised renal clearance of 2-butoxyacetic acid, the hemolytic metabolite, an increase in metabolism of 2-butoxyethanol to 2-buotoxyacetic acid, and a diminished degradation of 2-butoxyacetic acid to CO2 (Ghanayem et al., 1990
). These responses collectively lead to increased circulating levels of 2-butoxyacetic acid in blood and thus an increased potential for hemolysis. This, in turn, would result in increased levels of free iron, which would increase production of reactive oxygen species. Oxidative damage also increases with age, resulting either from increased free radical production or decreased antioxidant activity (Kaneko et al., 1997
). Additionally, repair of oxidative DNA damage is lower in older rodents (Fraga et al., 1990
). All of these pathways potentially impact on 2-butoxyethanol carcinogenesis. Additional studies are needed to examine the relationship of rodent age to 2-butoxyethanol-induced hepatic changes.
The species selectivity for the induction of oxidative stress by 2-butoxyethanol may be explained in part by differences in antioxidant levels between rodents. A decrease in vitamin E, a major antioxidant in the liver, was observed in both rat and mouse liver treated with 2-butoxyethanol. Although 2-butoxyethanol treatment reduced vitamin E levels in both rat and mouse liver, the basal level of vitamin E was approximately 2.5-fold higher in the rat compared with the mouse liver. The maximum reduction of vitamin E in rat liver by 2-butoxyethanol resulted in vitamin E levels that were higher than control vitamin E levels in mouse liver. A similar finding with hepatic vitamin E was reported in mice treated with dieldrin, a nongenotoxic and mouse-specific carcinogen (Bachowski et al., 1995; Klaunig et al., 1998
). In primary cultured mouse and rat hepatocytes, higher basal levels of vitamin E were also seen in rat hepatocytes, and rat hepatocytes were less sensitive to iron-induced oxidative stress and damage (Park et al., 2002b
). Therefore, the oxidative stress induced by 2-butoxyethanol selectively in the mouse may be related to lower basal levels of antioxidants (vitamin E).
Iron deposition, detected by Perl staining, was restricted to the Kupffer cell. Increased iron deposition may activate Kupffer cells, resulting in the production of a number of biologically active factors that may induce cell proliferation in endothelial cells and or hepatocytes. The induction of DNA synthesis by the peroxisome proliferators reportedly involves the activation of the Kupffer cell release of cytokines and growth regulatory molecules, at least in part (Rose et al., 1997, 1999
). In particular, TNF-
is believed to be involved in the mitogenic response of peroxisome proliferators (Bojes et al., 1997
; Rose et al., 1997
). In addition, Kupffer cell activation results in production of reactive oxygen species. Additional studies with peroxisome proliferators have shown an oxidant-dependent and species-specific activation of the transcription factor NF
B that correlated with hepatocellular proliferation (Rose et al., 1999
; Rusyn et al., 1998
). In addition to inducing DNA damage and lipid peroxidation, the production of reactive oxygen species, whether derived from Kupffer cell activation or other biological processes, can activate oncogenes (e.g., AP-1, Bcl-2, and myc) that can stimulate cell proliferation or inhibit apoptosis (Manna et al., 1998
; Müller et al., 1997
). Collectively, these findings support the proposal that Kupffer cell activation (induced by iron loading) and subsequent release of reactive oxygen species and cytokines are involved in the mechanism of 2-butoxyethanol-induced carcinogenesis. Further studies are needed to determine whether Kupffer cells are activated following 2-butoxyethanol treatment and whether expression of cell growth regulatory genes are altered after exposure to 2-butoxyethanol in the mouse.
This study showed that the mouse-specific tumorigenicity of 2-butoxyethanol may be related to the induction of cell proliferation/DNA synthesis secondary to oxidative stress, possibly mediated by Kupffer cell activation due to iron deposition from hemolysis (Fig. 1). Although hemolysis and subsequent iron deposition in the rat liver were observed, no oxidative damage or DNA synthesis was observed in the rat. The observation that basal vitamin E levels are substantially greater in the rat than in the mouse suggests that the rat is refractory to tumor induction by 2-butoxyethanol due, at least in part, to a higher antioxidant capability.
The above data support our proposed mode of action for mouse liver tumor induction by 2-butoxyethanol. Critical to this mode of action are the steps of iron overload via hemolysis induction by 2-butoxyacetic acid and the induction of oxidative damage (involving a depletion of antioxidants, i.e., vitamin E). In considering the relative human risk of 2-butoxyethanol exposure to liver cancer induction, it is important to note that these two steps of the mode of action are not apparently satisfied in humans. Comparative studies involving red blood cells have demonstrated that human red blood cells are refractory to the hemolytic effect of 2-butoxyacetic acid (Udden, 2000). Similarly, a reduction in antioxidant vitamin E levels in humans by 2-butoxyethanol may not be easily obtained, as the basal liver vitamin E levels in humans is approximately 100-fold higher than those seen in mouse liver (Rocchi et al., 1997
). As seen in this study, higher liver vitamin E levels in rat liver correlated with a lower level of oxidative damage and cell proliferation compared with mouse liver. Thus human liver, with substantially higher vitamin E levels and the lack of a hemosiderotic state, lacks the essential steps in the mode of action necessary for the induction of liver neoplasia.
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ACKNOWLEDGMENTS |
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NOTES |
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