Toxicological Sciences 65, 177-183 (2002)
Copyright © 2002 by the Society of Toxicology
CARCINOGENICITY |
Acrylamide-Induced Cellular Transformation
Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Dr., MS 1021, Indianapolis, Indiana 46202
Received June 7, 2001; accepted September 25, 2001
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ABSTRACT |
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Acrylamide is a monomer of polyacrylamide, whose products are used in biochemistry, the manufacture of paper, water treatment, and as a soil stabilizer. While polymeric acrylamide is nontoxic, the monomer can cause several toxic effects and has the potential for human occupational exposure. While acrylamide is not mutagenic in prokaryotic mutagenesis assays, chronic acrylamide treatment in rodents has been shown to produce tumors in both rats and mice. The mechanism for the induction of tumors by acrylamide is not known. In the present study, we examined the possibility that acrylamide might induce cellular transformation, using Syrian hamster embryo (SHE) cell morphological transformation as well as potential mechanisms for the cellular transformation. Results showed that treatment with 0.5 mM and higher concentrations of acrylamide continuously for 7 days induced morphological transformation. Cotreatment with acrylamide and N-acetyl-L-cysteine (NAC), a sulfhydryl group donor, resulted in the reduction of acrylamide-induced morphological transformation in SHE cells. Cotreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, and acrylamide produced no change in morphological transformation when compared to acrylamide treatment only. Cotreatment with acrylamide and DL-buthionone-[S,R]-sulfoximine (BSO), a selective inhibitor of
-glutamylcysteine synthetase, increased the percent of morphologically transformed colonies compared to acrylamide treatment alone. Acrylamide reduced GSH levels in SHE cells, and cotreatment with acrylamide and NAC prevented the acrylamide-induced reduction of GSH. BSO treatment with acrylamide enhanced the depletion of GSH. These results suggest that acrylamide itself, but not oxidative P450 metabolites of acrylamide appear to be involved in acrylamide-induced cellular transformation and that cellular thiol status (possibly GSH) is involved in acrylamide-induced morphological transformation. Key Words: acrylamide; carcinogenicity; oxidative stress; Syrian hamster embryo (SHE) cells; morphological transformation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acrylamide is a monomer of polyacrylamide, which is used in both industrial and laboratory processes. While polymeric forms of acrylamide are relatively nontoxic, the monomer hydrophilic forms are more toxic. A recent study reported that acrylamide was formed in the heating of rodent feed, suggesting that human exposure to acrylamide could occur during the cooking of rodent food (Tareke et al., 2000
). Chronic acrylamide treatment produced tumors in rats and mice. In these studies, acrylamide produced testicular mesotheliomas and thyroid tumors in male rats and thyroid, mammary, and central nervous system tumors in female rats (Friedman et al., 1995; Johnson et al., 1986; Dearfield et al., 1988). Acrylamide also induced skin neoplasia in SENCAR mice in the presence of the tumor promoter, TPA (Bull et al., 1984b), and lung tumors in SWISS-ICR mice (Bull et al., 1984a). The mechanism of the observed acrylamide carcinogenicity is unresolved.
Chromosomal aberrations and sister chromatid exchanges were observed in vitro with acrylamide treatment (Adler et al., 2000; Tsuda et al., 1993). Although acrylamide binds to DNA directly via a Michael addition reaction in vitro (Solomon et al., 1985) and in vivo (Segerbäck et al., 1995), acrylamide failed to produce mutagenicity in bacterial mutagenesis systems, either in the presence or in the absence of S9 mix for metabolic activation (Hashimoto and Tani, 1985).
Acrylamide appears to be metabolized into glycidamide via P450 2E1 (Calleman et al., 1990; Sumner et al., 1999), forming a DNA-reactive epoxide that implies genotoxicity (Segerbäck et al., 1995). However, the tissue and organ distribution of DNA-glycidamide adducts do not correlate with acrylamide-induced tumors in rat organs (Segerbäck et al., 1995). Additional studies have failed to show mutation activity by acrylamide except for clastogenic activity in germ cells (Hashimoto and Tani, 1985; Tsuda et al., 1993). Thus, it is possible that nongenotoxic or epigenetic mechanisms may participate in acrylamide-induced tumorigenicity in rodents. One possible epigenetic mode of action may be related to the affinity of acrylamide for sulfhydryl groups in the cell and in glutathione (GSH, the thiols of nonprotein), specifically. Acrylamide is detoxified by conjugation with GSH. This may result in a depletion of cellular GSH stores and result in a change in the redox status of the cell. This change, in turn, may modulate gene expression directly or via the transcription factors that are redox-regulated, and may lead to apoptosis, cell proliferation, or transformation (Abate et al., 1990; Schulze-Ostoff et al., 1995). Acrylamide also binds to cysteine residues of proteins (Hashimoto and Aldridge, 1970; Miller et al., 1982; Srivastava et al., 1986). Acrylamide reactivity with enzymes or receptors may induce changes in cellular functions and signal pathways, leading to a possible involvement with acrylamide-induced carcinogenesis. Changes in dopamine receptor affinity and alterations of thyroid stimulating hormones, prolactin, and testosterone levels have been observed in rats following acrylamide treatment (Agrawal et al., 1981; Ali et al., 1983). Prolonged hormonal derangement is associated with the induction of mutations and the carcinogenic process (Furth, 1975), and the tumors observed following chronic treatment were localized to endocrine-sensitive organs. Acrylamide also binds to cytoskeletal proteins (Hartley et al., 1997; Sickles et al., 1995). Disruption of the cytoskeletal system has been documented in neoplastic cells and reflects the altered shape and altered motility of the transformed cells (Pienta and Coffey, 1992). Cytoskeletal disruption by the carcinogenic process can alter growth-related cellular function (Liaw and Schwartz, 1993; Pienta and Coffey, 1992).
Morphological transformation of Syrian hamster embryo (SHE) cells mimics the early stage of carcinogenesis, while established cell lines BALB/c3T3 and C3H/10T1/2 cells are considered to represent later stages of the carcinogenesis process (Yamasaki et al., 1996). Acrylamide has previously been shown to induce morphological transformation in C3H/10T1/2 and NIH/3T3 cells (Banerjee and Segal, 1986) as well as in the BALB/c3T3 cell line (Tsuda et al., 1993). Transformation of SHE cells has been widely used in studies examining the mechanisms of chemical carcinogenesis (Barrett et al., 1984; Isfort et al., 1994). In the present study we specifically addressed whether: (1) acrylamide can induce transformation in SHE cells, (2) P450 metabolism of acrylamide was necessary to produce the cellular transformation, (3) modulation of GSH levels in SHE cells produced a change in cell transformation by acrylamide, and (4) possible interaction of 17-ß-estradiol on acrylamide-induced transformation (Kaster et al., 1997) could be observed upon further examination of preliminary findings.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Acrylamide (purity greater than 99.9%) was purchased from Midwest Scientific Co. (St Louis, MO). Benzo[a]pyrene, L-glutamine, dimethylsulfoxide(DMSO), N-acetyl-L-cysteine (NAC), 1-aminobenzotriazole (ABT), DL-buthionone-[S,R]-sulfoximine (BSO), and 17ß–estradiol (E2) were purchased from Sigma Chemical Co. (St Louis, MO). Test chemicals were either stored at 22°C (acrylamide), or 0–4°C (NAC, ABT, and BSO). Le Boeuf's Dulbecco's Modified Eagles Medium (DMEM-L) was from Quality Biological (Gaithersburg, OH). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). L-glutamate was purchased from Gibco (Grand Island, NY).
Cell culture.
SHE cells were isolated and cultured as described previously (Kerckaert et al., 1996). Briefly, primary embryo cells were isolated from 13-day gestation Syrian golden hamsters (Charles River, Wilmington, MA) and cryopreserved. The primary cells were grown in Le Boeuf's Dulbecco's modified eagle medium (pH 6.7), supplemented with 20% fetal bovine serum and 4 mM L-glutamate at 37°C in 10% CO2 at 90% relative humidity. A feeder layer was prepared by plating 2 x 106 SHE cells into 30 ml of complete media in a T-150 tissue culture flask. After a 48-h incubation, the cells were detached with 0.05% trypsin and 0.02% Na2EDTA in Ca+2-, Mg+2-free Hank's balanced salt solution (HBSS). The cells were irradiated (5000 rads) on ice for 40 min, and plated at a density of 4 x 104 cells/60-mm culture dish in 2 ml of medium. The target cells were prepared by plating 1 x 106 thawed cells per T-25 tissue culture flask containing 5 ml of complete media. After a 24-h incubation, the cells were detached with trypsin/Na2EDTA and plated onto the feeder layer at a density of 60–80 cells/60-mm culture dish with 2 ml of medium, giving a total volume of 4 ml. After a 24-h incubation, cells were continuously exposed to acrylamide, with and without the other test compounds. In addition, for NAC treatment of SHE cells, the pH was adjusted with 1 N NaOH to that of control media. Cells were treated with BSO for 24 h in advance and then coincubated with acrylamide for 6 days. At sampling, the cultures were then rinsed, fixed with methanol, stained with Giemsa (Sigma, St. Louis, MO), and evaluated for the presence of a morphologically transformed phenotype, using a Nikon stereoscopic zoom microscope. A morphologically transformed colony was defined as previously described (Kerckaert et al., 1996). For each group, total colony number, morphological transformation frequency [(the number of transformed colonies/total number of colonies scored) x 100], and the relative plating efficiency [RPE, (test group plating efficiency/solvent control plating efficiency) x 100] were determined.
Statistics.
Collected data were statistically analyzed by Fisher's exact test for morphological transformation studies. Treatment groups were considered statistically different at p < 0.05 (Armitage, 1971).
Measurement of GSH.
AM-treated cells were washed with PBS, collected by scraping from culture dishes, resuspended with buffer, and analyzed for glutathione (GSH) following the method of Harvey et al. (1989). The suspended cells were treated with perchloric acid (0.1 M) to precipitate proteins. After centrifugation, the supernatant was injected onto HPLC columns. Separation was achieved with 2 radial-pak liquid chromatograph cartridges (8 mm; Waters, Milford, MA) and a mobile phase (2% aqueous acetonitrile, 50 mM sodium phosphate monobasic, and 0.05 mM octanesulfonic acid, pH 3.4, adjusted by 85% phosphoric acid) at a flow rate of 1 ml/min. The GSH amount was measured by electrochemical detection (Coulochem 5200 with 5020 guard cell and 5010 analytical cell; esa, Chelmsford, MA) set at 400 mV potential, 0.5 µA range. GSH concentration was calculated from the GSH standard curve. Protein content of the extracts was determined using Bio-Rad DC protein assay kit that is based on the method of Lowry et al. (1951), with bovine serum albumin as a standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acrylamide cytotoxicity was examined in SHE cells at concentrations ranging from 0.01 to 10 mM after 7 days of continuous treatment. Cytotoxicity, measured by a reduction in relative plating efficiency (RPE), is shown in Table 1
. Cytotoxicity (50% or greater reduction in RPE) was observed at acrylamide concentrations of 1.0 mM and higher (Table 1). For the subsequent cell transformation assays, 0.1, 0.3, 0.5, or 0.7 mM acrylamide was used (Table 2). Cytotoxicity measurements of BSO, NAC, and ABT were performed, and the concentrations used for subsequent experiments were determined (highest subcytotoxic concentrations examined; data not shown).
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SHE cells were treated with Acrylamide for the morphological transformation study at concentrations ranging from 0.1 to 0.7 mM with 7 days of exposure (Table 2
). A dose-dependent increase (p < 0.05) in morphological transformation relative to control (media) was observed at 0.5 and 0.7 mM acrylamide (Table 2).
1-Aminobenzotriazole (ABT), a nonspecific suicidal P450 inhibitor, was used to block oxidative metabolism of acrylamide through cytochrome P450. The effect of ABT cotreatment with acrylamide on cell transformation is shown in Figure 1. ABT cotreatment with either 0.5 or 0.7 mM acrylamide produced no change in transformation frequency compared with acrylamide only. Treatment with ABT only had no effect on morphological transformation compared to media and DMSO control groups (Fig. 1).
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DL-Buthionone-[S,R]-sulfoximine (BSO), a selective inhibitor of
-glutamylcysteine synthetase and the rate-limiting step in GSH synthesis, was utilized to examine the role of GSH in acrylamide-induced SHE cell morphological transformation. The effect of BSO cotreatment with acrylamide on morphological transformation is shown in Figure 2. The concentrations of BSO examined (0.1, 1.0, and 5.0 µM) did not statistically increase morphological transformation in SHE cells compared with media and DMSO control groups. Cotreatment with BSO and either 0.3 or 0.5 mM acrylamide resulted in a significant increase in acrylamide-induced morphological transformation (Fig. 2). At the highest doses of both BSO (5.0 µM) and acrylamide (0.5 mM) tested, a decrease in relative plating efficiency (29.6%) was observed, suggestive of cytotoxicity (Fig. 2) at this dose combination.
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N-acetyl-L-cysteine (NAC), a thiol donor, was used to examine the effect of sulfhydryl supplementation on acrylamide-induced SHE cell morphological transformation. The effect of NAC cotreatment with acrylamide in cell transformation is shown in Figure 3
. Treatment with NAC (1.0 or 2.5 mM) alone had no effect on morphological transformation. Cotreatment with either NAC (1.0 or 2.5 mM) and acrylamide (0.3, 0.5, or 0.7 mM) resulted in a reduction of the morphological transformation frequency in SHE cells observed following treatment with acrylamide only. The rate of transformation in the NAC-treated groups was consistent with the transformation rate of untreated controls.
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The results of these experiments showed that maintenance of GSH levels appears to be involved in acrylamide-induced morphological transformation. Additional studies were performed to examine the effects of acrylamide, BSO, and NAC on GSH levels in SHE cells (Figs. 4 and 5
). Selected concentrations of BSO were used in cotreatment with 0.3 mM acrylamide for 5 and 24 h. Treatment with 5 µM BSO for 5 h reduced GSH to 65% of control while 24-h treatment reduced GSH to 35 and 5% of control level with 0.1 and 5.0 µM BSO, respectively. Cotreatment with 0.1, 1.0, and 5.0 µM BSO together with 0.3 mM acrylamide for 5 h reduced GSH levels to 81, 73, and 59% of control, respectively. A reduction in GSH levels to 20, 8, and 6% of control was seen following cotreatment with 0.1, 1.0, and 5.0 µM BSO together with 0.3 mM acrylamide, respectively, for 24 h (Fig. 4). NAC was used in cotreatment with 0.5 mM acrylamide for 5 h. Treatment with 0.5 mM acrylamide for 5 h reduced GSH to 75% of control level. Cotreatment with 0.5 mM acrylamide with either 1.0 or 2.5 µM NAC restored GSH to control levels (Fig. 5).
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Since the targets for neoplastic formation in the rat following acrylamide treatment are hormonal-sensitive sites, one hypothesis put forth by our group was that acrylamide modulates the hormonal milieu of the rat, resulting in an increase in cell proliferation in the target tissues, which, coupled with the other cellular effects of acrylamide, results in neoplastic development. Previously, we have reported an additive effect on SHE cell transformation following cotreatment with selected hormones together with acrylamide. These studies were performed with concentrations of acrylamide that did not induce morphological transformation. We further examined this effect of acrylamide, in concert with 17ß-estradiol, on SHE cell morphological transformation. 17ß-Estradiol treatment alone showed a tendency to increase morphological transformation frequency of SHE cells (p = 0.066), while cotreatment with 17ß-estradiol and 0.5 mM acrylamide induced a significant increase in morphological transformation frequency, which shows a higher-than-additive level of each treatment (Table 3
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cellular transformation has been used to detect and understand the mechanisms of genotoxic and nongenotoxic carcinogens (Barrett et al., 1984
; Isfort et al., 1994). The SHE cell transformation model mimics the multistage nature of carcinogenesis. In the present study, acrylamide treatment at concentrations of 0.5 mM or higher induced morphological transformation in SHE cells. This confirms results from previous transformation studies in other cell models including C3H/10T1/2, NIH/3T3, and BALB/c 3T3 cells (Banerjee and Segal, 1986; Tsuda et al., 1993). Acrylamide induced cellular transformation in these models at similar concentrations to that seen in the present study with SHE cells (0.2 mM–1.0 mM, depending on cell type).
Acrylamide has demonstrated clastogenic properties, having been shown to induce aneuploidy, chromosomal aberrations, and sister chromatid exchange in Chinese hamster V79 cells (Moore et al., 1987; Shiraishi, 1978; Tsuda et al., 1993). Other compounds including benzene, estrogen, and estrogen-like compounds that produce aneuploidy and have clastogenic activity in vitro have also demonstrated transformation activity in SHE cells (Gibson et al., 1995; Hayashi et al., 1996; Tsutsui and Barrett, 1997; Tsutsui et al., 1997).
Inhibition of acrylamide oxidative metabolism P450 enzymes by ABT treatment produced no change in SHE cell transformation frequency, suggesting that oxidative metabolite(s) of acrylamide is not involved in acrylamide-induced SHE cell transformation. Previous studies using cell lines have not addressed the role of metabolite(s) in cell transformation (Banerjee and Segal, 1986; Tsuda et al., 1993). In a previous study of DNA adduct formation in rodents following acrylamide treatment, the levels of DNA adducts with glycidamide, a DNA reactive epoxide metabolite, were measured. Glycidamide binds to cellular DNA and induces mutagenicity in the Ames test (Hashimoto and Tanii, 1985). In acrylamide-treated rodents, glycidamide DNA adduct formation was evenly distributed throughout the body in contrast to the observed organ pattern of acrylamide carcinogenicity (Segerbäck et al., 1995). This suggests that metabolism of acrylamide may not be necessary for DNA adduct formation and its carcinogenic effects and that the parent compound acrylamide may be involved in the carcinogenesis. In addition, the observed toxic effects of acrylamide in in vitro studies with Chinese hamster ovary cells and mouse lymphoma cells without exogenous metabolic activation suggest that metabolism is not involved in the transformational/carcinogenic effect (Tsuda et al., 1993; Moore et al., 1987). Our current results support this premise, in that inhibition of P450 metabolism by ABT failed to modify acrylamide-induced cell transformation in the SHE cells. In contrast to this, a recent in vivo study reported that ABT pretreatment inhibited or reduced the dominant lethal effect of acrylamide (125 mg/kg) in mice, suggesting that glycidamide, the epoxide metabolite of acrylamide is the cause of germ cell mutation in mouse spermatids (Adler et al., 2000). However, in that study it was also suggested that a chromosomal protein of sperm cells is alkylated directly by the parent acrylamide that may be the mechanism of acrylamide-induced clastogenicity. Conversion of acrylamide to glycidamide by cytochrome P450 enzymes is inversely related to the level of parent acrylamide in rats, as determined from hemoglobin adduct formation (Bergmark et al., 1991). Only 13% of parent compound is converted to glycidamide with a high dose (100 mg/kg) of acrylamide, while 50% is converted to glycidamide with a low dose (5 mg/ml).
The well established reactivity of acrylamide with proteins may also participate in the carcinogenesis process by modifying cellular functions and signal pathways. Acrylamide treatment has been shown to alter dopamine receptor affinity as well as changes in hormonal levels (Agrawal et al. 1981; Srivastava et al., 1986). These changes have been suggested as a mode of action in the induction of tunica vaginalis mesotheliomas in acrylamide-treated male Fisher rats. Acrylamide disruption of the cytoskeletal proteins may be involved in acrylamide-induced cellular transformation carcinogenicity. Microtubule disruption has been shown to change cellular response and to stimulate DNA synthesis of bovine endothelial cells (Liaw and Schwartz, 1993). Although cytoskleletal disruption was not examined in the present study, alteration in growth factors' responsiveness to mitogens, cell differentiation, and cytoskeleton has been reported in the SHE cell transformation process (Isfort et al., 1994). Also, alteration of growth-related cell function has been attributed to altered shape and altered motility in other transformed cells (Isfort et al., 1994; Pienta and Coffey, 1992). Acute exposure to acrylamide induced change in thyroid gland morphology in female Fisher rats (Khan et al., 1999).
Acrylamide has been shown to deplete GSH and inhibit gluthathione S-transferase activity in vitro and in vivo (Srivastava et al., 1986). In the present study, depletion of GSH with pretreatment of BSO-enhanced acrylamide induced transformation frequency. Acrylamide and glycidamide GSH conjugates have been detected in the urine of treated rats. A reduction of cellular GSH level by acrylamide treatment was observed in our study. However, the reduction of GSH levels by BSO treatment only did not result in transformation, suggesting that GSH depletion by itself was not sufficient to induce cell transformation. Therefore, acrylamide must have additional properties in the transformation process besides strictly GSH depletion. However, maintenance of GSH levels with NAC prevented the induction of acrylamide cell transformation supporting an important role for GSH in the acrylamide-induced transformation process.
17ß-Estradiol treatment alone showed a tendency to induce morphological transformation frequency in SHE cells (p = 0.066), while cotreatment with 17ß-estradiol and acrylamide induced a synergistic effect on the increase in morphological transformation frequency. Estrogen and estrogen-like chemicals are able to transform SHE cells (Barret et al., 1981; Metzler and Schiffmann, 1991; Tsutui et al., 1987). However, nonhormonal mechanisms are involved in estrogen-induced SHE cell morphological transformation, since there are no measurable levels of estrogen receptors in SHE cells (Korach and McLachlan, 1985) and no correlation between hormonal potency and the ability of SHE cell morphological transformation (McLachlan et al., 1982). Estrogen binding to tubulin (Epe et al., 1987), DNA adduct formation by estrogens and metabolites in vivo and in vitro (Cavalieri et al., 1997; Stack et al., 1996), and induction of aneuploidy (Tsutsui et al., 1983,1987) have been suggested as alternate mechanisms for cell transformation and carcinogenesis by 17ß-estradiol. These cellular effects by estrogens are similar to those seen with acrylamide.
In summary, acrylamide treatment for 7 days continuously induced morphological transformation in SHE cells. Cotreatment with 17ß-estradiol and acrylamide produced an additive effect on morphological transformation frequency in SHE cells. Acrylamide itself, but not an oxidative metabolite(s) appears to be involved in the SHE cell transformation. Decrease of GSH by acrylamide treatment or by BSO treatment played a role in acrylamide-induced transformation in SHE cells since conjugation of acrylamide to GSH is a detoxification pathway of acrylamide, protecting SHE cells from morphological transformation. Therefore, we propose that clastogenic activity of acrylamide as well as acrylamide reactivity to macromolecules leading to structural and cellular functional change via GSH depletion in SHE cells might be involved in acrylamide-induced SHE cell morphological transformation. Modification of GSH has been shown to participate in cell signaling, subsequently affecting and modifying gene expression (Allen and Tresini, 2000; Trosko et al., 1998). Further detailed examination of the role of GSH and GSH transferases would help further delineate the mechanisms of acrylamide-induced transformation/carcinogenicity.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (317) 274-7787. E-mail: jklauni{at}iupui.edu.
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