Acrylonitrile-induced morphological transformation in Syrian hamster embryo cells
Haizhou Zhang,
Lisa M. Kamendulis,
Jiazhong Jiang,
Yong Xu and
James E. Klaunig1
Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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Abstract
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Acrylonitrile (ACN) is a monomer used in the synthesis of rubber, fibers and plastics. Previous studies demonstrated that ACN induces brain neoplasms (predominately astrocytomas) in rats following chronic treatment. While the mechanisms of ACN-induced glial cell carcinogenicity have not been completely elucidated, investigations by our group and others have suggested a role for the induction of oxidative stress and the resultant oxidative damage in this process. In vitro cell transformation models are useful for detecting and studying the mechanisms of chemical carcinogenesis. Cell transformation by chemical carcinogens in Syrian hamster embryo (SHE) cells exhibits a multistage process similar to that observed in vivo, for both non-genotoxic and genotoxic carcinogens. In the present study, the ability of ACN to induce morphological transformation and oxidative damage was examined in SHE cells. ACN induced an increase in morphological transformation at doses of 50, 62.5 and 75 µg/ml (maximum sub-toxic dose tested) following 7 days of continuous treatment. SHE cells exposed to ACN for 24 h failed to increase morphological transformation. Morphological transformation by ACN was inhibited by co-treatment with the antioxidants
-tocopherol and ()-epigallocathechin-3 gallate (EGCG) for 7 days. Treatment of SHE cells with 75 µg/ml ACN produced a significant increase in 8-hydroxy-2'-deoxyguanosine that was also inhibited by co-treatment with
-tocopherol or EGCG. These results support the proposal that oxidative stress and the resulting oxidative damage is involved in ACN-induced carcinogenicity.
Abbreviations: ACN, acrylonitrile; B[a]P, benzo[a]pyrene; CEO, 2-cyanoethylene oxide; 2'-dG, 2'-deoxyguanosine; DMEM-L, DMEM LeBoeuf's modification; DMSO, dimethylsulfoxide; EGCG, ()-epigallocathechin-3 gallate; GSH, reduced glutathione; OH8dG, 8-hydroxy-2'-deoxyguanosine; ROS, reactive oxygen species; SHE, Syrian hamster embryo.
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Introduction
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Acrylonitrile (ACN) is a widely used intermediate in the manufacture of acrylic fibers, plastics, synthetic rubbers and resins (1,2). Human exposure to ACN could potentially occur during the manufacturing process, end-products usage, cigarette smoke and via contamination of drinking water (35). The acute toxic effects of ACN in rodents include CNS dysfunction, adrenal necrosis, lung edema and congestion and gastric bleeding (69). Chronic exposure of rats to ACN by gastric gavage, inhalation or in drinking water resulted in increased incidence of brain astrocytoma, Zymbal's gland carcinoma and forestomach and mammary gland tumors (1012). Among the observed neoplasms, induction of astrocytomas occurred with the highest incidence and consistency. In contrast to results seen in rats, human epidemiological studies have been negative for ACN-induced neoplasia (1316).
Depletion of cellular reduced glutathione (GSH), cyanide formation via ACN metabolism and covalent protein binding of ACN have been suggested as mechanisms for the observed acute toxic effects of ACN (6,1720). The mechanism(s) of ACN-induced carcinogenicity remains unresolved. However, previous studies by our group (21,22) and others (23) have shown that ACN exposure is associated with the induction of oxidative stress, indicated by oxidative damage to DNA, and a decrease in antioxidant enzyme activity, selectively in rat brain following in vivo exposure and in rat glial cells in vitro. Oxidative stress results when the cellular concentration of reactive oxygen species (ROS) exceeds the antioxidant (both enzymatic and non-enzymatic) capability of the cell (24). ROS are produced continuously as a result of both endogenous and exogenous functions. Damage resulting from excess oxygen radicals has been implicated in the pathogenesis of aging and several associated chronic diseases, including cardiovascular and neurodegenerative diseases (25,26), rheumatoid arthritis (27) and cancer (2830). With respect to carcinogenesis, ROS formation and the resulting oxidative damage have been associated with several steps in the multistage cancer process (28,29). Once generated, ROS can interact with and modify both structural and functional cellular macromolecules. The result ultimately is modification of cell function and/or cell death through oxidative damage to lipids (lipid peroxidation), DNA and/or proteins. Formation of DNA hydroxyl adducts can interfere with normal cell growth by causing genetic mutations and/or altering normal gene transcription. Oxidative DNA damage can lead to the formation of mutations through nucleotide modification, alterations in hydrogen bonds and conformational changes in the DNA templates (31). Of the oxidative DNA modifications, 8-hydroxy-2'-deoxyguanosine (OH8dG) is the most abundant oxidative DNA damage formed (32). Oxidation of fatty acids by ROS to lipid peroxyl radicals and lipid hydroperoxides can result in modification of cellular membrane structure and function, as well as formation of DNA adducts (33). ROS may also target proteins and result in stimulation or inhibition of enzyme activity, changes to receptor proteins and/or modification of membrane transport.
In order to further define the mechanism(s) by which ACN produces a carcinogenic response in rats, an in vitro transformation model utilizing Syrian hamster embryo (SHE) cells was employed. SHE cell transformation has been used to detect both genotoxic and non-genotoxic carcinogens and to further understand the mechanisms of the transformation process (34). In the present studies, ACN was examined for induction of morphological transformation and oxidative DNA damage (OH8dG) in SHE cells. In addition, the effect of antioxidants [
-tocopherol and ()-epigallocathechin-3 gallate (EGCG)] on ACN-induced morphological transformation and OH8dG formation was examined.
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Materials and methods
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Chemicals
ACN (>99% pure) was purchased from Aldrich Chemical Co. (Milwaukee, MI). Benzo[a]pyrene (B[a]P), L-glutamine, dimethylsulfoxide (DMSO),
-tocopherol acetate, citric acid and sodium acetate were purchased from Sigma Chemical Co. (St Louis, MO). EGCG was a gift from Dr Junshi Chen (Institute of Preventive Medicine, Chinese Academy of Science, People's Republic of China). The WB DNA extractor kit was purchased from Wako (Richmond, VA). DMEM LeBoeuf's modification (DMEM-L) was from Quality Biological (Gaithersburg, OH).
SHE cell transformation assay
The SHE cell transformation assay, performed at pH 6.7, was conducted as previously described (35). Briefly, primary embryo cells were isolated from female Syrian golden hamsters at day 13 of gestation (Harlan Sprague-Dawley, Indianapolis, IN) and cryopreserved. Feeder cells were prepared by propagating 2x106 cells in DMEM-L. Following 48 h incubation, the cells were detached, exposed to X-ray irradiation (~5000 rad) and plated (4x104 cells/60 mm culture dish). After a 24 h period, target cells were plated (80 cells/dish) onto the feeder layer. Following an additional 24 h, cell cultures were exposed to ACN for 1 day (followed by replacement with ACN-free medium for an additional 6 days) or 7 days continuously. In studies examining the effects of the antioxidants
-tocopherol and EGCG, ACN and either
-tocopherol (50150 µM) or EGCG (5 µM) were co-incubated for 7 days. Following either treatment protocol, SHE cell colonies were then fixed, stained and scored for morphological transformation after 7 days in culture. For each group, the total number of transformed and non-transformed colonies, transformation frequency [(no. of malignantly transformed colonies/total no. of colonies)x100] and relative plating efficiency [(treatment group total no. of colonies/solvent control total no. of colonies)x100] was measured. In all studies, B[a]P treatment at 10 µg/ml for 7 days was included as a positive control.
Analysis of OH8dG
For the analysis of OH8dG, SHE cells were plated at 1x106 in 100 mm culture dishes. Following an overnight incubation, cell cultures were exposed to ACN in the presence or absence of
-tocopherol or EGCG for 17 days. OH8dG was measured as described previously (36). Briefly, the DNA was isolated using the sodium iodide choatropic isolation methodology (37). Isolated DNA was dissolved in 10 mM TrisHCl buffer (pH 7.0) containing 2 mM butylated hydroxytoluene and 0.1 mM desferal. DNA (100200 µg) was sequentially digested with 10 U nuclease P1 (37°C, 30 min) and 14 U alkaline phosphatase (37°C, 60 min). After centrifugation (10 000 g, 10 min, 4°C), the supernatant was analyzed for OH8dG by HPLC with electrochemical detection (E1, 100 mV, 5 µA; E2, 350 mV, 2 µA; Coularray II; ESA, Chelmsford, MA). 2'-Deoxyguanosine (2'-dG) was analyzed by UV detection at 260 nm (Waters Photodiode Array Detector; Waters Inc., Milford, MA). OH8dG and 2'-dG were quantified from standard curves prepared on the day of analysis. Results are reported as the ratio of OH8dG to 2'-dG.
Statistics
Collected data were statistically analyzed by Fisher exact test for morphological transformation studies, one way ANOVA followed by Duncan's test for OH8dG studies. Treatment groups were considered statistically different at P < 0.05.
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Results
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Cytotoxicity
Initial studies were performed to examine the cytoxicity of ACN (measured by inhibition of relative colony number) in SHE cells after either 7 days of continuous treatment or 24 h of treatment. Cytotoxicity (50% reduction or greater in SHE cell colony number) was observed at ACN concentrations >75 µg/ml for the 7 day treatment (Table I
) and for the 24 h treatment (data not shown). For subsequent 7 day cell transformation assays, 75 µg/ml ACN was used as the highest sub-cytotoxic concentration of ACN. Cytotoxicity was also evaluated in SHE cells following treatment with
-tocopherol and EGCG (data not shown). Concentrations >150 µM
-tocopherol reduced SHE cell colony number. ECGC produced no observable cytotoxicity up to 2 mM (maximum dose tested). From previous in vitro studies (38), a concentration of 5.0 µM EGCG has been shown to produce maximal antioxidant effects and therefore this concentration of EGCG was used in subsequent experiments.
Morphological transformation by ACN
The effects of ACN on SHE cell transformation following 7 and 1 day treatments are shown in Tables II and III
, respectively. When SHE cells were treated with ACN at concentrations from 12.5 to 75 µg/ml for 7 days, the transformation frequency increased significantly in a dose-dependent manner at 50.0, 62.5 and 75 µg/ml (Table II
). Following a 24 h ACN treatment (and 6 days of ACN-free medium), no morphological transformation was observed at any ACN concentration examined (Table III
). For both exposure durations, B[a]P (a positive control) produced a statistically significant increase in transformation rate (1.77% for 7 day treatment and 1.13% for 24 h treatment) (Tables II and III
).
Effect of antioxidants on ACN-induced morphological transformation
As shown in Table II
, exposure to either 50 or 75 µg/ml ACN for 7 days produced a significant increase in morphological transformation. In a first set of experiments, the effect of a single concentration of
-tocopherol (100 µM) was examined for its effect at three concentrations of ACN. Co-treatment of either 25, 50 or 75 µg/ml ACN with 100 µM
-tocopherol for 7 days resulted in a reduction in the frequency of morphological transformation seen with ACN only treatment (Table IV
). The rate of transformation was reduced to untreated control levels. Treatment with
-tocopherol alone had no effect on morphological transformation.
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Table IV. Inhibition of ACN-induced morphological transformation by the antioxidant -tocopherol in SHE cells following a 7 day treatment
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In a second study, a single concentration of ACN co-treated with one of three concentrations of
-tocopherol was examined. Morphological transformation in SHE cells co-treated with 50 µg/ml ACN and either 50, 100 or 150 µM
-tocopherol for 7 days was reduced in an
-tocopherol concentration-related manner (Table V
).
-Tocopherol alone did not have any effect on morphological transformation in SHE cells.
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Table V. Inhibition of ACN-induced morphological transformation by various concentration of the antioxidant -tocopherol in SHE cells following a 7 day treatment
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The water-soluble antioxidant EGCG (from the water-soluble polyphenolic fraction of tea) was also examined for its effect on ACN-induced morphological transformation. EGCG by itself had no effect on morphological transformation. Morphological transformation produced by 50 and 75 µg/ml ACN was blocked by co-treatment with 5 µM EGCG for 7 days (Table VI
).
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Table VI. Inhibition of ACN-induced morphological transformation by the antioxidant EGCG in SHE cells following a 7 day treatment
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OH8dG induced by ACN in SHE cells
The above results support the proposition that oxidative stress is involved in the transformation process induced by ACN. Additional studies examined the induction of OH8dG by ACN and the effects of
-tocopherol and EGCG on oxidative DNA damage in SHE cells. Following treatment with 75 µg/ml ACN, OH8dG levels increased to 192 and 186% of control after treatment with ACN for 2 and 3 days but no increase was observed after 1 or 7 days (Figure 1
). Although elevated above control, no significant increase in OH8dG was observed in SHE cells treated with ACN for 1 and 7 days. Interestingly, the basal level of OH8dG in untreated control cells increased with time over the 7 day culture period.

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Fig. 1. Analysis of OH8dG in ACN-treated SHE cells. SHE cells were cultured in 100 mm cell culture dishes. At 75% confluency cell cultures were treated with 75 µg/ml ACN for 1, 2, 3 and 7 days. After treatment, DNA was isolated and OH8dG was determined by HPLC coupled with EC detection. Data are reported as means ± SD (n 4). *P < 0.05 versus control (ANOVA post-hoc Duncan's test).
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Since a significant increase in OH8dG was observed following either 2 or 3 days exposure to 75 µg/ml ACN, the effects of the antioxidants
-tocopherol and EGCG on ACN-induced OH8dG were examined at these two time points. OH8dG levels in SHE cell cultures treated with either
-tocopherol (100 µM) or EGCG (5 µM) were not different from untreated controls (Figure 2
). Importantly, the increase in OH8dG produced by 75 µg/ml ACN seen after 2 and 3 days treatment was reduced to control levels by treatment with 100 µM
-tocopherol or 5 µM EGCG (Figure 2
).
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Discussion
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The formation of morphologically transformed colonies is the first identifiable stage in the transformation process in SHE cells (39). In the present study, ACN produced an increase in transformation at doses of 50, 62.5, and 75 µg/ml following 7 days of continuous treatment (Table II
) but failed to produce transformation after a 24 h treatment (Table III
). The 7 day treatment results were in agreement with previously reported studies on cellular transformation in vitro by ACN (4043). Previous studies have not addressed short-term ACN exposure (24 h). Studies with the SHE transformation model have suggested that transformation-dependent treatment duration was indicative of the potential mode of action for the compound (35). These results support a non-direct DNA reactive mode of action for ACN, consistent with that seen previously with tumor-promoting agents (35). Reports on the DNA reactivity of ACN have been equivocal. While several studies have reported mutagenicity by ACN (44,45), other studies have shown a negative result in the presence or absence of an activation system (46,47). Importantly, when ACN has been studied in in vivo genotoxicity assays, including the chromosomal aberration, micronucleus test, sister chromatid exchange and dominant lethal assays, the findings have been negative (4852).
Although conjugation with GSH is a major elimination pathway for ACN, ~1015% of an administered dose of ACN is converted to 2-cyanoethylene oxide (CEO) by cytochrome P450 2E1 (5356). CEO, which is capable of covalent binding to nucleic acids (57,58), has been suggested to be a critical intermediate in ACN-induced carcinogenicity (59). CEO has been shown to be mutagenic in in vitro systems (60,61). Therefore, the results of the present study cannot rule out a possible role of CEO in the morphological transformation of SHE cells. However, target tissues (the brain) of ACN-induced carcinogenesis appear to have little metabolic capacity for enzymatic oxidation of ACN, while liver (not a target organ) has a high capacity for metabolic activation (5356). Furthermore, CEO does not appear to be preferentially taken up or retained in target organs of ACN carcinogenesis (62) and DNA adducts have not been found in rat brain following administration of ACN (57). Collectively, these data suggest that direct DNA damage by ACN or its metabolites are not involved or are of little impact on the induction of neoplasia by ACN in rat brain.
Oxidative stress, arising from a redox shift towards the production of ROS relative to antioxidants, has been linked to the pathogenesis of a variety of diseases, including cardiovascular and neurodegenerative diseases and cancer. With respect to ACN, the formation of ROS and subsequent oxidative damage may be produced through several pathways. Conjugation with GSH, an important cellular antioxidant, is a major route of detoxification of ACN. ACN treatment results in an early depletion of GSH (21,22,56,6365) that may contribute to an overall decrease in cellular antioxidant content leading to the induction of oxidative stress. Additionally, cyanide appears to be liberated during ACN metabolism. Cyanide has also been linked to the induction of oxidative stress both through ROS production (inhibition of the mitochondrial respiratory chain) and inhibition of catalase. Finally, ROS may be produced as a by-product of ACN metabolism via P450 2E1 oxidation or through futile cycling (66).
Additional studies have provided data supporting ROS formation and subsequent induction of oxidative stress by ACN. Ivanov et al. showed that daily i.p. administration of 40 mg/kg ACN to rats for 2 or 4 weeks led to increased exhalation of ethane, an index of lipid peroxidation (67). The increased ethane formation was prevented by vitamin E co-treatment (67). ACN was also shown to potentiate ROS production and oxidative stress in rat alveolar macrophages (68). Duverger-van Bogaert et al. noted that the indirect mutagenesis due to ACN was mediated by a radical-type reaction (69).
In the present study, an involvement of oxidative stress in morphological transformation by ACN was supported by several observations. OH8dG, a biomarker of oxidative DNA damage, is formed by the reaction of ROS with DNA (34,70,71). While a statistically significant increase in OH8dG was only seen after 2 and 3 days of treatment, the levels of OH8dG formation were elevated at all sampling times (Figure 1
). The lack of a statistically significant increase in OH8dG formation in SHE cells after 1 day of exposure is consistent with the failure of 24 h ACN exposure to induce morphological transformation. OH8dG is mutagenic (7274), causing GC
TA transversions during DNA replication. Thus, the early increase in OH8dG produced by ACN may have been sufficient to induce the genetic events necessary for morphological transformation in SHE cells. Concomitant with an increase in oxidized DNA (OH8dG), co-treatment of ACN with the lipid-soluble antioxidant
-tocopherol and water-soluble antioxidant EGCG resulted in a decrease in both transformation (Tables IVVI

) and formation of oxidized DNA damage (Figure 2
). The level of OH8dG in untreated SHE cell cultures was also shown to increase over time (Figure 1
). As demonstrated by Liehr, cells maintained in culture may be stressed by exposure to an artificially high oxygen environment compared with that observed in vivo (75). In addition, given the fact that the culture medium was not replaced during the 7 day culture period, antioxidants may be exhausted during the assay, contributing to the oxidative stress. Thus, culture conditions may have contributed to the increase in the basal level of OH8dG observed.
Excess production of reactive oxygen intermediates has been shown to modulate gene expression (76). In particular, ROS formation has been shown to activate the transcription factors NF-
B and AP-1 (77). These factors may be of importance in both cellular transformation and chemical carcinogenesis as they play a central role in the regulation of genes involved in cellular defense mechanisms and cellular growth regulation. In addition to being activated by ROS, NF-
B activation can be blocked by antioxidants such as vitamin E (78).
In summary, ACN induced morphological transformation and oxidized DNA (OH8dG) in SHE cells following 7 days exposure that was blocked by co-treatment with antioxidants. Accumulating evidence points to oxidative stress as a mechanism involved in ACN-induced morphological transformation and carcinogenicity. The induction of oxidative stress by ACN appears to involve depletion of cellular antioxidants and possibly DNA repair enzymes. Additional studies aimed at examining the role of ACN metabolism, in particular the production of CEO and cyanide and the potential for ROS generation during oxidative metabolism, may provide insights into the mechanisms involved in the cell transformation and carcinogenesis processes.
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Acknowledgments
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Supported, in part, by the AN Group, Inc.
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Notes
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1 To whom correspondence should be addressed Email: jklauni{at}iupui.edu 
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Received August 3, 1999;
revised October 13, 1999;
accepted November 8, 1999.