Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, MS 1021, 635 Barnhill Drive, Indianapolis, Indiana 46202
Received December 12, 2001; accepted February 8, 2002
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ABSTRACT |
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Key Words: acrylonitrile; catalase; glutathione; oxidative stress; SHE cells; superoxide dismutase; xanthine oxidase.
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INTRODUCTION |
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While the mechanism(s) by which acrylonitrile induces carcinogenicity in rats is unclear, previous studies by our group and others have shown that acrylonitrile treatment results in the selective induction of oxidative stress in rat brain in vivo and rat glial cells in vitro (Jiang et al., 1998; Kamendulis et al., 1999
; Whysner et al., 1998
). In addition, in Syrian Hamster Embryo (SHE) cells, an in vitro cell transformation model that correlates with carcinogenesis in vivo, acrylonitrile increased oxidative DNA damage (8-hydroxydeoxyguanosine; OH8dG) and morphological transformation (Zhang et al., 2000
). Both OH8dG formation and morphological transformation by acrylonitrile were inhibited by antioxidant cotreatment (Zhang et al., 2000
). These results support the involvement of oxidative stress in acrylonitrile-induced transformation/carcinogenesis. However, the mechanism(s) by which acrylonitrile induces oxidative stress remain unresolved.
Oxidative stress arises when the balance between reactive oxygen species (superoxide, hydroxyl radical, hydrogen peroxide, and singlet oxygen) and antioxidants favors the former (Sies, 1991). Reactive oxygen species are produced continuously as a result of both endogenous and exogenous processes. Once generated, reactive oxygen species 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 lipid (lipid peroxidation), DNA, and/or protein. To maintain cellular homeostasis, aerobic organisms, including humans, possess several enzymatic and nonenzymatic antioxidants that serve to counter reactive oxygen species (Janssen et al., 1993
; Yu, 1994
). Overproduction and/or inadequate removal of cellular oxidants due to deficiency of antioxidant content can result in oxidative stress. The resulting, unrepaired oxidative damage from oxidative stress has been suggested to participate in the pathogenesis of aging and several chronic diseases, including cardiovascular and neurodegenerative diseases (Giacosa and Filiberti, 1996
), rheumatoid arthritis (Mapp et al., 1995
), and cancer (Guyton and Kensler, 1993
; Trush and Kensler, 1991
). With respect to carcinogenesis, oxidative stress and the resulting oxidative damage have been linked to all stages of carcinogenesis (Guyton and Kensler, 1993
; Marnett, 1987
). As it was previously shown that the acrylonitrile-induced oxidative stress was associated with morphological transformation of SHE cells (Zhang et al., 2000
), the current studies examined potential mechanism(s) by which acrylonitrile induces oxidative stress. Specifically, studies examined the induction of reactive oxygen species and activity of several antioxidants following treatment with acrylonitrile in SHE cells, as well as determined the role of oxidative metabolism on acrylonitrile-induced morphological transformation and antioxidant activity.
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MATERIALS AND METHODS |
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Cell transformation.
The SHE cell transformation assay was performed as previously described (Kerckaert et al., 1996). Cryopreserved primary embryo cells from Syrian hamsters (SHE cells) were reconstituted and cultured for 72 h. Cells were irradiated (5000 rad), and plated at 4.0 x 104 cells per 60 mm culture dish. Target cells were then prepared by plating nonirradiated SHE cells at a density of 7090 target cells per dish over the existing cells. Cell cultures were treated following 24 h of incubation and allowed to incubate with acrylonitrile for 1 to 7 days. After the specified incubation times, medium was replaced with medium lacking acrylonitrile and cells were allowed to incubate to complete a 7-day period. Colonies were then fixed with methanol and stained with Geimsa. All studies included a positive control of 10 µg/ml B(a)P along with nontreated and solvent-treated (DMSO) control groups. Treatment groups were scored for total colony number, morphologically transformed (MT) colonies, transformation frequency (number of MT colonies/total number of colonies x 100), and plating efficiency (number of colonies per dish/number of target cells seeded x 100; Kerckaert et al., 1996
).
Cell culture and treatment.
Primary embryo cells were isolated from the embryos of Syrian golden hamsters at the 13th day of gestation (Harlan-Sprague-Dawley, Indianapolis, IN) and cryopreserved. For each experiment, SHE cells were prepared by propagating 2 x 106 cells in DMEM-L. The cells were cultured at 37°C and 10% CO2 and 95% humidity. At 90% confluency, cells were trypsinized and replated at a density of 1 x 106 cells per 60 mm culture dish. Following overnight incubation (approximately 75% confluency), cells were treated with 0, 25, 50, and 75 µg/ml of acrylonitrile for 4, 24, and 48 h. Although cell transformation was examined following treatment with acrylonitrile for 1 to 7 days, mechanistic studies were conducted following 4, 24, or 48 h of exposure.
Detection of hydroxyl radicals.
The level of hydroxyl radicals was determined as an index of reactive oxygen species formation in SHE cells. Hydroxyl radical formation was determined using an assay that measures the aromatic hydroxylation of salicylic acid (Floyd et al., 1984). Briefly, 4 h before harvest, SHE cells were loaded with 10 mM salicylic acid. After specified time intervals, the reaction was terminated by the addition trichloroacetic acid (5% final concentration), and the cells incubated on ice for 15 min. Cell extracts were centrifuged (10,000 x g, 10 min) and the resultant supernatant injected into HPLC. 2,3-DHBA was detected electrochemically (Colouchem II detector, set at 0.5µA, 350mV; ESA Inc., Chelmsford, MA), salicylic acid by UV detection (Waters 996 photodiode array set at 296 nm, Waters Inc., Milford, MA) and quantitated from standard curves. Hydroxyl radical formation was expressed as the ratio of 2,3-DHBA to salicylate and was used as an index of reactive oxygen species formation.
Measurement of glutathione (GSH).
After treatment, SHE cells were removed from the culture dishes and suspended in saline. Protein was precipitated with perchloric acid (10% v/v), and cell extracts obtained following centrifugation (5000 x g, 5 min). GSH was analyzed using HPLC with electrochemical detection (Colouchem 5200 with 5020 guard cell and 5010 analytical cell; ESA, Chelmsford, MA; set at +400 mV, 0.5 µA) as previously described (Harvey et al., 1989) and quantified from a standard curve. Perchloric acid-insoluble pellets were dissolved in 0.1 M NaOH (Bhave et al., 1996
) and protein content of the extracts determined, based on the method of Lowry et al. (1951), using bovine serum albumin as a standard (Bio-Rad DC protein assay kit).
Quantitation of antioxidant enzyme activity.
Following treatment, SHE cells were washed (PBS; 2x) and collected. 0.1% Triton X-100 in 10 mM potassium phosphate buffer (pH 7.2) was used to resuspend the cell pellets. Cells membranes were disrupted by sonication (1 cycle of 10 s; Fisher 550 sonic dismembranetor, Fisher Scientific, Pittsburgh, PA). The lysates were centrifuged (13,000 x g; 15 min), and the supernatants removed for enzyme measurement. Protein content of the supernatants was determined, based on the method of Lowry et al. (1951), using bovine serum albumin as a standard (Bio-Rad DC protein assay kit).
Superoxide dismutase activity was measured using the method of Marklund and Marklund (1974) based on the inhibition of the auto-oxidation of pyrogallol. Briefly, an aliquot of cell extract was mixed with Tris-cacodylic buffer (50mM Tris HCl, 50 mM cacodylic acid, 1mM diethylenetriamine pentaacetic acid, pH 8.2), and 2mM pyrogallol. The auto-oxidation of pyrogallol and the inhibition of this reaction were monitored spectrophotometrically. Under the conditions of this assay, 1 unit of superoxide dismutase activity is equivalent to the amount of enzyme that produces a 50% inhibition of the auto-oxidation of pyrogallol.
Catalase activity was analyzed according to the method of Cohen et al. (1970) by monitoring the enzyme-catalyzed decomposition of hydrogen peroxide using potassium permanganate. Fifty µl sample was added to a test tube. H2O2 was then added to the tube and incubated on ice for 3 min. H2SO4 was used to stop the reaction. Finally, KMnO4 solution was added and absorbance recorded at 480 nm. In this assay, 1 unit of enzyme activity equals k/(0.00693) (Aebi, 1974), where k = log (S0/S2) x (2.3/t), S0 = absorbance of standard absorbance of blank, S2 = absorbance of standard absorbance of sample, and t = time interval. The measured activities were normalized with the protein content of each sample.
Quantitation of xanthine oxidase activity.
Xanthine oxidase activity in treated SHE cells was measured by monitoring the enzyme-catalyzed formation of uric acid as described (Hashimoto, 1974). Treated SHE cells were trypsinized and centrifuged (2000 rpm, 5 min). Cell pellets were resuspended in sucrose (1 ml, 0.25M) and centrifuged (2000 rpm, 4 min). The resulting cell pellets were resuspended in sucrose (4 volumes, 0.25M). Membranes were disrupted by sonication on ice (1 cycle of 10 s; Fisher 550 sonic dismembranetor, Fisher Scientific, Pittsburgh, PA). Sixty µl of sample was added to 900 µl of reaction buffer (50 mM Na2HPO4, 0.1 mM potassium oxonate, 0.067mM xanthine sodium salt, pH 7.5) and allowed to react at 30°C for 30 min. The reaction was terminated using TCA (30 µl of 100%). Samples were centrifuged (10,000 x g, 10 min, 4°C) and uric acid quantitated by measuring the absorbance at 292 nm against a blank (reaction mixture without xanthine). Xanthine oxidase activity was calculated using the molar extinction coefficient of uric acid (7.6 x 103; Hashimoto, 1974
). One unit of xanthine oxidase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol of uric acid from xanthine per min at 30°C. Activity was expressed as U/mg protein. Protein concentration was determined (Bio-Rad DC protein assay kit), based on the method of Lowry et al. (1951), using bovine serum albumin as a standard.
Statistics.
Transformation was considered significant at p < 0.05, by a one-tailed Fisher's exact test conducted on pooled data (n = 40 dishes) from two or more trials (Armitage, 1971). All other data were analyzed by one-way ANOVA followed by Duncan's test. Treatment groups were considered statistically different at p < 0.05.
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RESULTS |
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DISCUSSION |
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Induction of oxidative stress and the resulting damage to critical cellular macromolecules can occur through an increase in the formation of reactive oxygen species by the compound overriding the antioxidant protection system or through a decrease in the antioxidants, allowing for an increase in the endogenously generated reactive oxygen species, or a combination of both effects. The present results showed that induction of oxidative stress by acrylonitrile appears to produce an early and temporal depletion of the cellular antioxidants glutathione, catalase, and superoxide dismutase. This inhibition of cellular antioxidants may account for the increase in reactive oxygen species observed following treatment with acrylonitrile for 4 h. After 24 h of treatment with acrylonitrile, superoxide dismutase activity returned to control level, while catalase activity and GSH levels were increased over control. The production of hydroxyl radicals by acrylonitrile, however, remained constant over the treatment duration examined, suggesting that the formation of hydroxyl radicals may involve other mechanisms besides antioxidant depletion.
Another source of hydroxyl radicals may be through increased xanthine oxidase activity. In the current studies, acrylonitrile increased the activity of this enzyme following treatment for 24 or 48 h. Xanthine oxidase uses oxygen as an electron acceptor to oxidize hypoxanthine or xanthine resulting in the generation of superoxide radical and hydrogen peroxide (Nishino et al., 1997). These compounds can undergo chemical reactions in biological systems to yield hydroxyl radicals. Although catalase activity and GSH levels were increased at 24 and 48 h, the concomitant increase in xanthine oxidase may have resulted in the increased hydroxyl radicals seen at these time points.
The induction of cellular transformation required at least 2 days of continuous treatment with acrylonitrile (Fig. 1). The observed continuous production of reactive oxygen species by acrylonitrile, resulting from an early temporal decrease in cellular antioxidant followed by an increase in xanthine oxidase activity and/or other mechanisms, may contribute to the transformation process. This is further supported by studies that showed increased oxidative DNA damage (OH8dG) after treatment with acrylonitrile for 48 h that was blocked by coincubation with antioxidants (Zhang et al., 2000
). Similarly, previous studies by our group showed an increase in transformation concomitant with an increase in OH8dG following photoactivated methylene blue reactive oxygen species production. These two findings support the association between oxidative DNA damage and cell transformation.
These present results also demonstrated an apparent requirement of P450 metabolism for acrylonitrile to produce its effects on morphological transformation and inhibition of catalase and superoxide dismutase in SHE cells. Without metabolism, acrylonitrile had no effect on purified catalase or superoxide dismutase activity. However, in the presence of metabolic sources (SHE cell homogenate), acrylonitrile produced a time dependent inhibition of catalase and superoxide dismutase activities. Similarly, the inhibition of oxidative metabolism of acrylonitrile by 1-aminobenzotriazole (ABT, a suicide inhibitor of cytochromes P450) attenuated the effects of acrylonitrile on morphological transformation and catalase. ABT treatment also inhibited the acrylonitrile-induced increase in xanthine oxidase activity. Collectively, these results suggest that the production of reactive oxygen species requires oxidative metabolism of acrylonitrile and/or a metabolite of acrylonitrile is mediating the effects on antioxidants.
Conjugation with glutathione is the major elimination pathway for acrylonitrile, while approximately 1015% of administered dose of acrylonitrile is converted to 2-cyanoethylene oxide by cytochrome P450 (Abreu and Ahmed, 1980; Guengerich et al., 1991
; Roberts et al., 1991
). Further metabolism of cyanoethylene oxide produces number of metabolites, including cyanide (Fennell et al., 1991
; Geiger et al., 1983
; Tardif et al., 1987
). Cyanide has been shown to generate reactive oxygen species in several in vitro systems (Ardelt et al., 1994
; Arai et al., 1999
; Gunasekar et al., 1996
; Kanthasamy et al., 1997
) and inhibits the activities of catalase, and glutathione peroxidase in the brain (Ardelt et al., 1989
). The observations showing that acrylonitrile-induced inhibition of catalase activity in SHE cells was prevented when oxidative metabolism was blocked leads to the speculation that cyanide may account for the effects observed on catalase activity.
Xanthine oxidase exists primarily as xanthine dehydrogenase (NAD+ dependent type) in vivo (Parks and Granger, 1986). The oxidation of hypoxanthine or xanthine catalyzed by xanthine dehydrogenase uses NAD+ as an electron acceptor; therefore, reactive oxygen species are not normally generated during oxidation. However, under conditions of ischemia and hypoxia (Sussman and Bulkley, 1990
), an increased conversion from the dehydrogenase form of the enzyme to the oxidase form occurs. Cyanide has been reported to produce hypoxia by inhibition of cytochrome oxidase (Way, 1984
). The increased xanthine oxidase activity seen after 24 and 48 h may be a result of the increased conversion of xanthine dehydrogenase to xanthine oxidase by cyanide-induced hypoxia. This concept is supported by the findings that inhibition of acrylonitrile metabolism by cotreatment with ABT abolished the effect of acrylonitrile on xanthine oxidase activity in SHE cells. Additional studies are necessary to confirm whether cyanide is responsible for the induction of xanthine oxidase. In addition, low levels of reactive oxygen intermediates have been shown to modulate gene expression (Schulze-Osthoff et al., 1995
). In particular, reactive oxygen species are known activators of the transcription factors NFkB and AP-1 (Angel and Karin, 1991
). 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. Up-regulation of xanthine oxidase gene expression has been observed following treatment with agents that produce reactive oxygen species (Pfeffer et al., 1994
), supporting a role for reactive oxygen species in the regulation of this enzyme.
The increased GSH levels and catalase activity seen in SHE cells treated with acrylonitrile for 24 and 48 h may also result from reactive oxygen species induced alteration of gene regulation. The synthesis of GSH involves enzymatic conjugation of cysteine and glutamic acid by -glutamylcysteine synthetase, followed by peptide bond formation with glycine by glutathione synthetase. Bond formation by
-glutamylcysteine synthetase is the rate-limiting step in GSH synthesis (Lu, 1998
). Oxidative stress has been shown to activate AP-1 that subsequently up-regulates
-glutamylcysteine synthetase (Morales et al., 1998
). Expression of the catalase gene appears to be up-regulated by hydrogen peroxide and other agents that generate reactive oxygen species (Cornelissen et al., 1997
; Rohrdanz and Kahl, 1998
). While AP-1 and NFkB binding sites have been identified in the 5` flanking region of the catalase gene in the rat, these transcription factors may not be important in the regulation of catalase gene expression (Van Remmen et al., 1998
). In addition, catalase is regulated both at the posttranscriptional and posttranslational level (Reimer et al., 1994
), as well as through gene amplification (Yamada et al., 1991
). Further studies are needed to determine whether the acrylonitrile-induced increased xanthine oxidase and catalase activities and cellular GSH status are regulated at the transcriptional, posttranscriptional, or posttranslational level.
Based on the data presented herein, the induction of oxidative stress and cellular transformation by acrylonitrile in SHE cells appears to involve a temporal decrease in antioxidants (enzymatic and nonenzymatic) and activation of an oxidant enzyme (xanthine oxidase). The potential role of cyanide and other metabolites of acrylonitrile in the inhibition of cellular antioxidants was suggested since oxidative metabolism by cytochrome P450 was required for acrylonitrile to produce effects on catalase, superoxide dismutase, and xanthine oxidase. In addition, the elevation in reactive oxygen species seen following exposure to acrylonitrile may activate transcription factors that regulate the expression of xanthine oxidase, catalase, and/or enzymes related to GSH synthesis. These mechanisms may be acting in concert to induce oxidative stress and cellular transformation by acrylonitrile in SHE cells.
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NOTES |
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