Induction of oxidative stress and oxidative damage in rat glial cells by acrylonitrile
L.M. Kamendulis,
J. Jiang,
Y. Xu and
J.E. Klaunig1
Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS 1021, Indianapolis, IN 46202-5120, USA
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Abstract
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Chronic treatment of rats with acrylonitrile (ACN) resulted in a dose-related increase in glial cell tumors (astrocytomas). While the exact mechanism(s) for ACN-induced carcinogenicity remains unresolved, non-genotoxic and possibly tumor promotion modes of action appear to be involved in the induction of glial tumors. Recent studies have shown that ACN induced oxidative stress selectively in rat brain in a dose-responsive manner. The present study examined the ability of ACN to induce oxidative stress in a rat glial cell line, a target tissue, and in cultured rat hepatocytes, a non-target tissue of ACN carcinogenicity. Glial cells and hepatocytes were treated for 1, 4 and 24 h with sublethal concentrations of ACN. ACN induced an increase in oxidative DNA damage, as evidenced by increased production of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in glial cells but not in rat hepatocytes. Hydroxyl radical formation following ACN treatment was also selectively increased in glial cells. Following 1 and 4 h of ACN exposure, the levels of the non-enzymatic antioxidant glutathione, as well as the activities of the enzymatic antioxidants catalase and superoxide dismutase were significantly decreased in the rat glial cells. Lipid peroxidation and the activity of glutathione peroxidase were not affected by ACN treatment in rat glial cells. No changes in any of these biomarkers of oxidative stress were observed in hepatocytes treated with ACN. These data indicate that ACN selectively induced oxidative stress in rat glial cells.
Abbreviations: 2,3-DHBA, 2,3-dihydroxybenzoic acid; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; ACN, acrylonitrile; dG, 2'-deoxyguanosine; GSH, glutathione; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; OTC, 2-oxothiazolidine-4-carboxylic acid; ROS, reactive oxygen species; SA, salicylic acid; SOD, superoxide dismutase
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Introduction
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Acrylonitrile (2-propenenitrile; ACN), used in the manufacture of acrylics, plastics and synthetic rubber causes a dose-related increase in glial cell tumors (astrocytomas) in rat brain following chronic inhalation or oral administration (1,2). The mechanism(s) by which ACN produces astrocytomas in rodents remains unclear. In vivo and in vitro genotoxicity studies on ACN have yielded inconclusive results (36). Studies examining DNA binding and adduct formation have shown a low level of DNA binding in rat liver DNA but no adducts have been identified in rat brain or other tumor target organs following ACN treatment (4). Since ACN induces tumors in the brain but not in the liver, DNA adduct formation by ACN does not appear to be the mechanism for astrocytoma formation. Based on these experimental findings, a non-genotoxic mode of action has been suggested for ACN carcinogenicity.
The induction of oxidative stress has been suggested as a possible mechanism of non-genotoxic chemical carcinogenesis and has been shown to participate in all of the stages of the carcinogenesis process, including initiation, promotion and progression (79). A role for the participation of oxidative stress in the astrocytoma formation by ACN is supported by several observations. Glutathione (GSH) conjugation, a major pathway of ACN metabolism (10), has been shown to be depleted following ACN treatment in vivo (11) and may decrease the antioxidant capacity of the cells resulting in an overall increase of intracellular reactive oxygen species (ROS) and oxidative damage. Metabolism of ACN results in the production of cyanide (3,4). Cyanide has been shown to induce oxidative stress (lipid peroxidation) in the brain of acutely treated mice and in cell lines (1214). Thus, the oxygen radicals produced may lead to the formation of oxidative DNA damage. In addition, due to a high oxidative capacity and a lowered antioxidant defense capacity relative to other organs, the brain may be more susceptible to oxidative damage (15,16). Recently, we and others have shown a selective and dose-dependent increase in oxidative damage by ACN in the rat brain (11,17). Therefore, the formation of ACN-induced astrocytomas in rats appears to be associated with an increase in oxidative stress in the brain.
The brain contains a mixed population of cell types, including astrocytes, the target of ACN carcinogenicity. A previously reported in vivo study demonstrating the selective induction of oxidative stress by ACN in the rat brain could not establish whether oxidative stress was produced specifically in the astrocyte (11). Thus, the purpose of the present investigation was to determine if ACN caused oxidative stress in rat astrocytes and to further define the mechanism for the induction of oxidative stress by ACN in rat astrocytes (an ACN target cell in vivo) and primary cultured rat hepatocytes (a non-ACN target cell in vivo). As primary cultured astrocytes represent a heterogeneous population of cell types, the DITNC1 cell line was utilized for these experiments. The DITNC1 astrocyte cell line was established from primary cultures of type 1 astrocytes from rat brain diencephalon. These cells retain characteristics consistent with the phenotype of type 1 astrocytes including immunoreactivity towards glial fibrillary acidic protein, and an uptake mechanism for
-aminobutyric acid (18). For oxidative stress measurements, several biological endpoints were measured. Oxidative DNA damage was examined by 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation, lipid damage was estimated by malondialdehyde (MDA) formation, a product of lipid peroxidation, and hydroxyl radial generation was determined by the aromatic hydroxylation of salicylic acid (SA) to 2,3-dihydroxybenzoic acid (2,3-DHBA) (19). Additionally, the non-enzymatic antioxidant, GSH and the enzymatic antioxidants, superoxide dismutase (SOD) catalase, and glutathione peroxidase (GSH-Px) were measured.
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Materials and methods
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Chemicals
ACN (>99% purity) was purchased from Aldrich (Milwaukee, WI). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). Dulbecco's modified Eagles's medium/Hamm's F-12 (DMEM/F12) and all other reagents were from Sigma (St Louis, MO).
DITNC1 astrocyte culture
The DITNC1 rat astrocyte cell line (18) was obtained from American Type Culture Collection (ATCC; Rockville, MD). Cells were grown in DMEM/F12 medium containing 4.5 g/l glucose, 10% FBS and 10 ml penicillinstreptomycin solution. Cell cultures were incubated at 37°C and 5% CO2 and media changed at 48 h during propagation of the cell line. All cells used for these studies were between passages 12 and 14 after receipt and were incubated at 37°C, 5% CO2. Twenty-four hours prior to treatment with test chemicals, cells were plated at a density of 1x106 in 60 mm culture dishes. For studies examining the effects of 2-oxothiazolidine-4-carboxylic acid (OTC) and vitamin E on acrylonitrile-induced oxidative stress, astrocytes were treated with the test chemicals simultaneously for a 24 h period.
Hepatocyte isolation and cell culture
Rat hepatocytes were isolated by two-step in situ collagenase perfusion as described previously (20). Following perfusion, the cells were filtered and centrifuged (twice at 300 g, 3 min). Viability, determined by trypan blue exclusion, was routinely >90%. Cells (1x106) were plated on 60 mm culture dishes in DMEM/F12 medium containing insulin (5 µg/ml), gentamycin sulfate (50 mg/ml), dexamethasone (0.8 µg/ml) and 5% FBS, and cultured in an atmosphere of 5% CO2, 37°C and 95% humidity. Medium was changed after 4 h. ACN was added to culture dishes following incubation overnight.
Assessment of cytotoxicity
Cytotoxicity of ACN was determined by lactate dehydrogenase (LDH) leakage after treatment for up to 24 h. LDH release was analyzed in culture media removed after 1, 4 or 24 h of ACN treatment and total LDH was analyzed in cells using a COBAS Mira S clinical analyzer with Sigma LD-L reagent. The percentage of total LDH released into cell culture medium was reported.
DNA isolation and analysis of 8-OH-dG
DNA was isolated by the method of Marmur (21) with modifications, for the analysis of 8-OH-dG (22). Briefly, 5x106 cells were homogenized in lysis buffer (10 mM TrisHCl, 1 mM EDTA, 1% SDS, pH 7.0). The homogenate was digested sequentially with proteinase K (10 U, 30 min, 37°C) and RNase A (5 U, 10 min, 37°C). Following each digestion, the solution was precipitated with ice-cold isopropanol and centrifuged (12 000 g, 15 min). The DNA samples were dissolved in 200 µl of 10 mM TrisHCl and digested with nuclease P1 (10 U, 30 min, 37°C) and alkaline phosphatase (14 U, 60 min, 37°C). After centrifugation (12 000 g, 10 min), 1.0 ml of supernatant was used for HPLC analysis. Elution was with a mobile phase consisting of 100 mM sodium citrate (pH 5.2) at a flow rate of 1.0 ml/min using a Waters Nova-Pak C18 reversed-phase analytical column. 8-OH-dG was detected electrochemically (Colouchem 5200 with 5020 guard cell and 5010 analytical cell; set at +350 mV potential, 0.5 µA range, esa; Chelmsford, MA). 2'-Deoxyguanosine (dG) was detected at 250 nm (Waters 996 system; Waters, Milford, MA). 8-OH-dG and dG were quantitated from standards prepared in mobile phase immediately prior to sample analysis.
Analysis of lipid peroxidation
The lipid peroxidation product MDA was measured as described (23). Briefly, 5x106 cells were homogenized in 1.0 ml of 0.1 M perchloric acid and 2 mM EDTA. The homogenate was centrifuged (5000 g, 4°C, 5 min) and the supernatant derivatized with 2,4-dinitrophenyl hydrazine (DNPH, 15.65 mM, 60 min, 25°C) and extracted twice with pentane (200 µl). The extracts were pooled, dried under nitrogen, and reconstituted in mobile phase (49% acetonitrile). The MDAhydrazine derivative was analyzed by UV-HPLC at 330 nm (Waters 484 system; Waters, Milford, MA). MDA concentration was calculated from MDA standards prepared immediately prior to analysis.
Assay for hydroxyl radicals
Levels of ROS, 2,3-DHBA, a non-enzymatic hydroxylation product of SA, were measured by HPLC-EC using the method of Floyd with modifications (24). Cells were co-treated with 1 mM SA (pH 7.3) and ACN. After the specified incubation periods, the cells were lysed with trichloroacetic acid (0.5 vol, 10%). The samples were centrifuged (12 000 g, 10 min) and the supernatants injected on HPLC. 2,3-DHBA formation was analyzed by electrochemical detection (Coulouchem 5200, 5020 guard cell and 5010 analytical cell, set at +350 mV potential, 0.5 µA range; esa). SA was detected by absorbance at 298 nm (Waters 996 system). The concentration of 2,3-DHBA and SA was calculated from matrix derived standard curves. The ratio of 2,3-DHBA to SA was compared among samples to reflect ROS production.
Measurement of GSH
GSH was analyzed by HPLC-EC as described previously (25). For GSH analysis, cells were washed twice with PBS (0°C), scraped from culture dishes and suspended in saline (0.9%). Protein was precipitated with perchloric acid (0.1 M), followed by centrifugation (5000 g, 5 min.). The supernatants were immediately injected onto HPLC columns. GSH content was measured by electrochemical detection (Colouchem 5200, 5020 guard cell and 5010 analytical cell, set at +400 mV potential, 0.5 µA range; ESA, Chelmsford, MA). GSH concentration was calculated from GSH standards prepared immediately prior to analysis.
Protein determination
For protein determination, cells were washed twice in PBS (0°C), scraped and the pellets were resuspended in 0.1% Triton X-100 containing 1.0 mM potassium phosphate buffer (pH 7.2). Cell homogenates were sonicated three times (10s; 550 sonic dismembraneator; Fisher Scientific, Pittsburgh, PA). The samples were centrifuged (13 000 g; 15 min), and the protein content of the supernatants determined using Bio-Rad DC protein assay kit (Bio-rad, Hercules, CA), based on the method of Lowry (26), with bovine serum albumin as a standard.
Assay of catalase activity
Catalase activity was measured by monitoring enzyme-catalyzed decomposition of H2O2 (27). Briefly, a solution of H2O2 was added to test tubes containing samples, a water blank, and an H2O2 standard solution (standard). After a 3 min incubation, the enzymatic reaction was terminated by the addition of H2SO4. KMnO4 (1.4 ml) was added to each tube, vortexed and absorbance recorded at 480 nm. One unit of catalase activity is defined as k/(0.00693) (28), where k = log (So/S2)x(2.3/t), So is absorbance of standard minus absorbance of blank, S2 is absorbance of standard minus absorbance of sample and t is time interval.
Measurement of SOD
SOD activity was measured by the inhibition of pyrogallol auto-oxidation (29). Briefly, 200 µl of cell extract was mixed with 750 µl cacodylic buffer (50 mM TrisHCl, 50 mM cacodylic acid, 1 mM diethylenetriamine pentacetic acid, pH 8.2) and 200 µl pyrogallol (2 mM). Absorbance was monitored at 420 nm for 3 min. One unit of SOD activity was defined as the amount of enzyme required to produce a 50% inhibition of pyrogallol autooxidation.
Assay of GSH-Px activity
GSH-Px activity was measured as described (30). An aliquot of 100 µl cell lysate was mixed with 800 µl reaction buffer (1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH and 1 mM GSH in PBS) and 100 µl (10 U) glutathione reductase. The mixture was vortexed and incubated (25°C, 5 min). GSH-Px activity was monitored at 340 nm and quantified from the amount of NADPH oxidized/min/mg protein.
Statistical analysis
The mean ± SD was determined for each treatment group. For each experimental endpoint, group differences (P < 0.05) relative to control were determined by one-way ANOVA followed by Dunnett's post-hoc analysis at each time point. Two-way ANOVA followed by Dunnett's post-hoc analysis was used to describe statistical differences (P < 0.05) across treatments and time points (31).
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Results
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The cytolethality of ACN was examined in both DINTC1 rat astrocytes (Table I
) and primary cultured rat hepatocytes (Table II
) using the measurement of the release of lactate dehydrogenase into cell culture medium. Cells were treated with 010 mM ACN for 1, 4 and 24 h. After 1 h, LDH was significantly elevated over control at concentrations of 5 mM and higher. After 4 and 24 h, cytolethality was observed in both cell types at
1.0 mM ACN. Morphological changes in cellular appearance were not observed at ACN concentrations of
1.0 mM at either 4 or 24 h. For subsequent experiments, sublethal concentrations of ACN (
1.0 mM) were used for both hepatocytes and astrocytes.
8-OH-dG levels were measured in DITNC1 astrocytes and rat hepatocytes (Table III
). In rat astrocytes, an increase in 8-OH-dG was observed and ranged from 1.9- to 3.8-fold over control after 4 h and 1.6- to 3.9-fold after 24 h. In rat hepatocytes, no increase in 8-OH-dG was observed at any concentration or time examined. The effect of ACN on induction of 8-OH-dG was reversible in rat astrocytes following removal of the compound (Table IV
). Following treatment with ACN for 24 h and removal for 4 h, 8-OH-dG levels returned to control levels in all but the 1.0 mM ACN group, in which a 32% reduction was observed. At 24 h following ACN withdrawal, 8-OH-dG levels had returned to control values at all concentrations examined (Table IV
).
The generation of ROS was examined in astrocytes and hepatocytes (Table V
). Intercellular ROS production, measured by the non-enzymatic hydroxylation of SA by the hydroxyl free radical, produces 2,3-DBHA. Following ACN treatment, an increase in 2,3-DHBA formation was seen (2- to 2.6-fold) after 4 and 24 h at ACN concentrations of 0.1 and 1.0 mM. ACN did not induce an increase in 2,3-DHBA formation in rat hepatocytes at any exposure duration or concentration examined (Table V
).
Oxidative lipid damage was evaluated in rat astrocytes and hepatocytes (Table VI
) by the detection of free MDA, a product of lipid peroxidation. No significant change in MDA levels was found in either cell type following treatment with ACN at any time or concentration evaluated.
Since no oxidative damage or free radical production was observed in rat hepatocytes following treatment with ACN, subsequent studies examined oxidative stress effects of ACN only in the rat glial cells. Levels of the cellular antioxidants, GSH (Table VII
), catalase, SOD and GSH-Px were evaluated (Table VIII
). Following exposure to 0.1 and 1.0 ACN, a significant decrease in GSH was observed in rat glial cells at 4 (2536% of control) and 24 h (4361% of control). Catalase activity showed no significant decrease from the control following ACN treatment in rat astrocytes at 4 and 24 h. A significant decrease in SOD activity was observed in rat astrocytes treated for 4 h with 1.0 mM ACN (39% reduction over control), and at 0.1 and 1.0 mM ACN in cells treated for 24 h (3840% reduction over control). In contrast to GSH and SOD, ACN treatment did not cause a significant change in GSH-Px activity in rat astrocytes.
Additional mechanistic experiments examined the effect of OTC, a precursor for GSH biosynthesis on ACN-induced production of ROS and 8-OH-dG in DITNC1 cells (Table IX
). OTC co-treatment was protective against the ROS generation and oxidative DNA damage produced by ACN. OTC co-treatment with ACN reduced 8-OH-dG formation produced by 0.1 mM ACN to control levels and by 66% in the 1.0 mM ACN treatment group (Table IX
). Similarly, ACN-induced hydroxyl radical formation, as evidenced by 2,3-DHBA formation, was significantly lowered (31 and 34% reduction with 0.1 and 1.0 mM ACN, respectively) in the presence of OTC. The effect of the antioxidant, vitamin E was also examined on ROS and 8-OH-dG formation in rat astrocytes (Table X
). Vitamin E co-treatment significantly reduced both ROS and 8-OH-dG formation caused by 0.1 and 1.0 mM ACN.
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Discussion
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In the present study, ACN induced oxidative stress in a tissue-specific manner. Significant increases in oxidized DNA (8-OH-dG) and ROS (hydroxyl radical) were observed selectively in a rat astrocyte cell line, whereas no increases in these parameters were seen in rat hepatocytes. DITNC1 cells, the cell line used for these studies, resemble primary type 1 astrocytes in that they retain reactivity towards glial fibrillary acidic protein and possess uptake mechanisms for GABA (18). The present results from a selective tumor target cell type, are in agreement with a previously reported in vivo study in which enhanced formation of these oxidative products was found elevated selectively in the rat brain cortex (11). Taken together, these findings correlate with the previously reported target tissue specificity for ACN tumorigenicity in the rat where the brain was the target tumor site.
The rat brain has been shown to have a lowered antioxidant capacity relative to the liver (11,16). Thus, rat astrocytes (the present study) may be susceptible to ACN-induced oxidative stress due to an inability to efficiently combat the oxidative insult produced by ACN. This hypothesis is supported by studies in cultured astrocytes that showed that depletion of GSH, an event that occurs following ACN treatment, results in enhanced oxidative damage (32). The selective increase in ROS by ACN (as indicated by 2,3-DHBA) in the astrocytes compared with ACN-treated hepatocytes together with the lowered antioxidant capacity in rat brain, may be acting in concert to produce the selective oxidative damage in astrocytes.
The mechanism by which the oxidative damage seen in the glial cells following ACN treatment results in tumor formation in the brain remains unresolved. The formation of ROS following chemical carcinogen treatment has been proposed to act on either or both the initiation and promotion stages of the cancer process (7,8). Oxidative DNA damage has been associated with the formation of intiated cells (7). 8-OH-dG formation can be induced by carcinogens including chemicals, metals and radiation and during aging (3335), and has been shown to correlate with the carcinogenic potential of a number of chemical agents (34,36). Additionally, 8-OH-dG has been shown to directly cause GC
TA and AT
GC transversions in DNA (37). Thus, 8-OH-dG produced following ACN exposure may participate in the observed carcinogenic actions of ACN through direct or indirect mechanisms. Although 8-OH-dG is an abundant DNA modification that results from oxidative insult, other DNA bases are also targets susceptible to oxidative modification. Therefore, additional DNA damage cannot be discounted and may be contributory to the observed carcinogenicity process. It is important to note that in the present study total cellular DNA (nuclear and mitochondrial) was measured in the ACN treated glial and liver cells. Therefore, the relative contributions of both of these sources of DNA to the observed 8-OH-dG cannot be defined at this time.
Oxidative damage and the formation of ROS have also been shown to participate in the tumor promotion process (7,8,38). In this study, the formation of 8-OH-dG and hydroxyl radicals observed following ACN treatment was temporal, dose-dependent and reversible following removal of the agent. These are well established properties of tumor promoting agents. A similar effect of ACN on another cellular mechanism associated with tumor promotion, inhibition of gap junctional intercellular communication, was also seen in rat glial cells (39). Given the lack of ACN mutagenicity and direct interaction with cellular DNA, it appears that ACN-induced astrocytomas in the rat are produced through non-genotoxic mechanisms involving tumor promotion.
The formation of hydroxyl free radicals was detected by the formation of 2,3-DHBA following SA treatment. ACN showed a dose-dependent increase in 2,3-DHBA levels in astrocytes and not in hepatocytes at any time point tested. Importantly, the formation of hydroxyl radicals by ACN correlated with the increased 8-OH-dG levels observed in astrocytes. In the liver, a non-target organ of ACN carcinogenicity, no increase in either hydroxyl radical (2,3-DHBA) or 8-OH-dG formation was observed, further supporting the target organ specificity of ACN-induced oxidative stress.
Lipid peroxidation (MDA) has been associated with aging, mutagenesis and carcinogenesis (40,41). In the present study, ACN failed to increase MDA in the glial cells at any concentration or time point examined. The lack of an increase in MDA is consistent with previous in vivo studies in which only small increases in MDA were observed during ACN exposure (11).
GSH, an intracellular peptide found in nearly all cell types (42), functions as an antioxidant to detoxify a variety of endogenously and exogenously generated free radicals. Decreased intracellular GSH levels have been implicated in the pathogenesis of a number of degenerative conditions and diseases including cancer (42). Depletion of GSH in cells therefore renders them more susceptible to oxidative damage. The astrocytes contain the major pool of GSH in the brain (43). However, ACN produced a concentration-dependent reduction in GSH levels in astrocytes, but not in rat hepatocytes at the time points examined. These results are in contrast with an in vivo study that showed that ACN treatment did not result in a significant depletion of GSH in rat brain and, therefore, was discounted as being involved in the formation of 8-OH-dG (17). In another in vivo study, ACN treatment, at doses that induced astrocytomas in the rat, produced an early depletion of GSH in the brain (11). The discrepancy between these two in vivo studies may be due, in part, to the time of sampling after ACN treatment, since GSH can readily cross the bloodbrain barrier, thus allowing for the compensation of depleted GSH in rat brain. In the present study, co-treatment of astrocytes with either OTC, a precursor to GSH biosynthesis, or with vitamin E, an antioxidant, significantly reduced 8-OH-dG and ROS generation induced by ACN treatment. These findings indicate that a number of factors are involved in the induction of oxidative stress by ACN and that cellular depletion of GSH may be important in the formation of oxidative stress.
Enzymatic antioxidants are another important group of cellular constituents responsible for maintaining the homeostatic balance between oxidants and antioxidants within a cell. SOD, catalase and Glu-Px, intracellular enzymatic antioxidants responsible for the removal of ROS such as H2O2 and superoxide free radicals were monitored. Previous studies have shown that basal activities of these enzymes are lower in the brain cortex than in the liver of rats (11,16). ACN lowered the activities of catalase and SOD in astrocytes. The mechanism(s) by which ACN decrease the activities of these enzymes are not known. It has been speculated that ACN may bind to, and thus inactivate, these enzymes. Due to the lowered relative capacities of the enzymatic antioxidants in the brain, astrocytes may innately be more susceptible to oxidative damage.
An additional mechanism for the generation of ROS by ACN is through the production of cyanide, produced by metabolism of ACN (4). Cyanide is known to uncouple oxidative phosphorylation and has been shown to produce oxidative stress in PC12 cells and lipid peroxidation in the brains of mice following in vivo treatment (1214). Important to the observation of induction of oxidative stress by ACN, the glial cell has been identified as a specific target of the deleterious actions of cyanide (44). Additionally, inhibition of cytochrome oxidase by ACN-induced hypoxia results in ROS formation (35). Thus, the contribution of cyanide to the induction of oxidative stress and glial cell tumorigenesis following ACN treatment needs to be evaluated further.
While it is apparent that ACN induces oxidative stress in a dose-dependent manner in rat brain (target tissue) and rat astrocytes (target cell type), the mechanism for this induction and the role of oxidative stress in ACN carcinogenesis remains unresolved. The production of ROS and the resulting oxidative DNA damage may involve one or more of the following processes: (i) direct generation of free radicals by ACN or a metabolite; (ii) stimulation of endogenously produced ROS by cytochrome P450 during ACN metabolism; (iii) direct binding of ACN (or a metabolite) to enzymatic and/or non-enzymatic antioxidants; (iv) interference with electron flow through respiratory chain in the mitochondria through the inhibitory actions of cyanide; or (v) altered gene expression through induction of gene transcription factors (i.e. Nf
B or AP1) or change in methylation status by ROS.
In summary, the current studies show that ACN treatment produced a concentration-dependent increase in oxidative stress in DITNC1 astrocytes, a target cell of ACN carcinogenicity. Oxidative stress was evidenced by increased formation of hydroxyl radical and formation of 8-OH-dG and a decrease in cellular GSH content and activities of SOD and catalase. In rat hepatocytes, these parameters were unaffected. These results, in concert with results from previous studies (11,39), support the formation of oxidative stress and oxidative DNA adduct formation as a mode of action of ACN carcinogenicity.
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Acknowledgments
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This study was supported in part by the Acrylonitrile Group Inc.
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Notes
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1 To whom correspondence should be addressedEmail: jklauni{at}iupui.edu 
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Received November 18, 1998;
revised March 24, 1999;
accepted April 12, 1999.