ToxSci Advance Access originally published online on June 2, 2006
Toxicological Sciences 2006 93(1):172-179; doi:10.1093/toxsci/kfl031
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Potential Role for Oxidative Stress in 2,2'-Dichlorobiphenyl–Induced Inhibition of Uterine Contractions but not Myometrial Gap Junctions
Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-2029
2 To whom correspondence should be addressed at Department of Environmental Health Sciences, University of Michigan, 109 Observatory Street, Ann Arbor, MI 48109-2029. Fax: (734) 763-8095. E-mail: rlc{at}umich.edu.
Received May 4, 2006; accepted June 1, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we reported that 2,2'-dichlorobiphenyl (2,2'-DCB)–induced decreases of amplitude and synchronization of uterine contractions are dependent on MAPK-induced phosphorylation of Connexin43 (Cx43) and inhibition of myometrial gap junctions. Recent studies show that oxidative stress inhibits uterine contractions and myometrial gap junctions also. The present study examines the hypothesis that 2,2'-DCB–induced modification of uterine contraction is dependent on oxidative stress–mediated inhibition of myometrial gap junctions via activation of mitogen-activated protein kinase (MAPK) and phosphorylation of Cx43. In uterine strips treated with
-tocopherol (100µM), deferoxamine mesylate (Def, 50µM), or superoxide dismutase (SOD, 1000 U) after a 1-h exposure to 100µM 2,2'-DCB, modification of uterine contractions reversed 1 h after initiating antioxidant treatment. Treatment of uterine strips with 100µM 2,2'-DCB for 1 h lowered total SOD activity and also induced a surge of superoxide generation after 5 min of exposure. However, myometrial cells exposed in culture to 100µM 2,2'-DCB did not produce reactive oxygen species as determined by the lack of superoxide anion generation measured by the cytochrome c reduction assay, reactive species by the formazan assay, hydrogen peroxide by the 2',7'-dichlorofluorescein assay, and lipid peroxidation by the thiobarbituric acid–reactive substance assay. Furthermore, cotreatment with SOD or Def was unable to prevent 2,2'-DCB–induced phosphorylation of Cx43, activation of MAPK, and inhibition of myometrial gap junctions. Although antioxidants reversed 2,2'-DCB–induced inhibition of uterine contraction force and synchronization, the myometrial cell culture experiments failed to support oxidative stress as a mechanistic link between 2,2'-DCB–induced inhibition of myometrial gap junctions and modification of uterine contraction. Key Words: 2,2'-dichlorobiphenyl; oxidative stress; uterine contraction; polychlorinated biphenyl; gap junctions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polychlorinated biphenyls (PCBs) are industrial compounds detected as contaminants in the air, water, sediments, and fish and in human adipose tissue, milk and, serum (Erikson, 1986; Safe et al., 1987
). There are 209 possible PCB congeners due to variations in the number and position of chlorine substitutions on the phenyl rings. It is known that meta/para-substituted coplanar PCB congeners, whose configuration is similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin, produce Ah receptor–mediated toxicity, whereas ortho-substituted noncoplanar PCB congeners show a different spectrum of toxicity independent of Ah receptor mechanisms (Brower et al., 1999; van den Berg et al., 1998).
There have been many reports that PCBs induce oxidative stress, defined as a disturbance in the equilibrium status of prooxidant/antioxidant systems in intact cells. For example, the PCB mixture Aroclor 1254 increases production of superoxide anion in brain and liver tissues (Hassoun et al., 2002), and the coplanar PCB congener 3,3',4,4',5-pentachlorobiphenyl increases generation of reactive oxygen species (ROS) in cultured rat cerebellar granule cells as shown by increased 2',7'-dichlorofluorescein (DCF) fluorescence (Lee and Opanashuk, 2004). Also, the noncoplanar, ortho-substituted PCB congener 2,2'-dichlorobiphenyl (2,2'-DCB) activates respiratory burst and produces ROS in human granulocytes as measured by luminol-amplified chemiluminescence (Voie et al., 1998) and stimulates ROS generation in rat synaptosomes as indicated by DCF fluorescence (Voie and Fonnum, 2000). Moreover, oxidative stress has been noted as a potential mechanism for endothelial barrier dysfunction induced by 3,3',4,4'-tetrachlorobiphenyl (Slim et al., 1999) and embryotoxicity induced by 3,3',4,4',5-pentachlorobiphenyl (Jin et al., 2001).
Krieger and Loch-Caruso (2001) showed that 
-hexachlorocylohexane (lindane) inhibits rat uterine contractions in vitro through an oxidative stress–mediated mechanism that likely involves inhibition of myometrial gap junctions. Gap junctions are cell membrane structures comprised of membrane-spanning aqueous channels. The gap junction channel consists of six proteins called connexins, and a large family of connexins has been identified (Saez et al., 2003). The cells of the smooth muscle layer of the uterus, the myometrium, become extensively coupled by gap junctions during parturition, preceded by increased expression of GJA1, also known as Connexin43 (Cx43) (Loch-Caruso, 1999). During parturition, the gap junction network allows the uterus to contract in a synchronous manner with increased force by allowing the rapid cell-to-cell dissemination of contraction-regulating chemical signals via the gap junction channels. Antioxidants protect inhibition of gap junction communication by motorcycle exhaust particles (Kuo et al., 1998) and by lindane in rat myometrial cells (Krieger and Loch-Caruso, 2001).
Previously, we reported that uterine strips from gestation day (GD) 10–pregnant rats exposed in vitro to the PCB congener 2,2'-DCB exhibit decreased contraction amplitude and decreased synchronization of contractions. Furthermore, we found that 2,2'-DCB–induced decreases in uterine contraction amplitude and synchronization are mediated through MAPK-mediated phosphorylation of Cx43 and inhibition of myometrial gap junctions. The present study investigates the hypothesis that 2,2'-DCB–induced modification of uterine contraction is dependent on oxidative stress–mediated inhibition of myometrial gap junctions via activation of MAPK and phosphorylation of Cx43. Although the results support oxidative stress as a potential mechanism for 2,2'-DCB–induced modification of uterine contractions, the data suggest that the oxidative stress–mediated effects on uterine contraction are independent of inhibition of myometrial gap junctions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Time-pregnant Sprague-Dawley rats were obtained from Harlan Laboratory (Indianapolis, IN). The animals were housed at ambient temperature (24 ± 1°C) under a 12-h light schedule. Animals used in this study were at GD 10.
Chemicals.
The 2,2'-DCB was purchased from Ultra Scientific (North Kingstown, RI). -Tocopherol, deferoxamine mesylate (Def), and superoxide dismutase (SOD) were purchased from Sigma (St Louis, MO).
Uterine contractility.
Spontaneous isometric oscillatory contractions were assessed in uterine muscle strips isolated from pregnant rats and suspended in standard muscle baths. Midgestation (day 10) Sprague-Dawley rats were anesthetized with ether, and the uteri were removed. Uterine strips 1 mm wide x 20 mm long were excised from the midsection of each uterine horn and suspended in 50-ml muscle baths filled with 37°C prewarmed physiologic salt solution (PSS: 116mM NaCl, 4.6mM KCl, 1.16mM NaH2PO4·H2O, 1.16mM MgSO4·7H2O, 21.9mM NaHCO3, 1.8mM CaCl·2H2O, 11.6mM dextrose, and 0.03mM CaNa2EDTA at pH 7.4). One end of each uterine strip was tied to a stationary post, and the other end was tied to a force transducer. The changes of contraction were recorded by computer using the PowerLab system (ADInstruments, Castle Hill, Australia). All strips were subjected to a 1.0-g preload tension, allowed to equilibrate for 45 min, and then depolarized with a brief exposure to 60mM KCl to determine maximal contractile force. Strips were rinsed free of KCl and allowed to equilibrate for 3 to 7 h until regular spontaneous oscillatory contractions were established. Contraction amplitude was calculated as the average peak force of contraction for the last 10 min of each 30- or 60-min exposure interval. Contraction completion was calculated as the number of peak force displacements that returned to baseline divided by the number of total contraction peaks in the last 10 min of each 30- or 60-min exposure interval. As such, contraction completion served as a measure of synchronization and regularity of the contraction pattern.
Cytochrome c reduction assay.
Quantification of superoxide production by cultured myometrial cells was based upon the method of Boota et al. (1997). Briefly, cells were removed from flasks, plated at 5500 cells/cm2, and incubated for 24 h at 37°C to allow for attachment and growth. Cells were then rinsed with phosphate-buffered saline (0.9mM CaCl2, 2.68mM KCl, 1.47mM K3PO4 [monobasic], 0.5mM MgCl2 [hexahydrate], and 8mM Na3PO4 [dibasic heptahydrate] at pH 7.2), and 1 ml of reaction buffer (2mM glucose, 1mM CaCl2, 1.3mM MgCl2, 4mM KCl, 100mM NaCl, and 10mM NaH2PO4(H2O), pH 7.4) was added to the dishes. Cytochrome c was added at a final concentration of 70µM, and Cu, Zn-SOD was simultaneously added to half the plates at 40 µg/ml final concentration. The reaction was stimulated by adding solvent (control) or 2,2'-DCB. After 5, 15, 30, or 60 min, the reaction was stopped by transferring aliquots of supernatant to microfuge tubes containing 25 µl of 1mM N-ethylmaleimide per 200-µl sample supernatant. Absorbance was then read at 550 nm, and superoxide production was determined based upon the difference in cytochrome c reduction with or without SOD. An extinction coefficient of 21.2 mM–1cm–1 was used for calculations. For the experiments with cell cultures, the results were expressed as nanomoles superoxide released per incubation time per 1 x 105 cells. For experiments with uterine tissue, 5-µg equivalent of membrane protein and 100-µg equivalent cytosolic protein were used.
SOD activity assay.
Uterine strips 1 mm wide x 20 mm long with endometrium and decidua were excised from the midsection of each uterine horn of GD 10 rats. They were treated with 100µM 2,2'-DCB for 1 h in 1-ml centrifuge tubes filled with 37°C prewarmed PSS and homogenized in cold 20mM HEPES buffer (pH 7.2) containing 1mM EGTA, 210mM mannitol, and 70mM sucrose per gram tissue. Homogenates were centrifuged at 1500 x g for 5 min at 4°C, and supernatants were used for SOD activity assay. Total SOD activity was measured by an SOD assay kit (Cayman Chemical, Ann Arbor, MI).
Myometrial cell isolation and culture.
Myometrial smooth muscle cells were isolated from midgestation Sprague-Dawley rats that had been anesthetized with ether and killed by cardiac puncture. Upon removal, uteri were immediately placed in ice-cold, calcium/magnesium-free phosphate-buffered saline (CMF-PBS: 2.68mM KCl, 1.5mM K3PO4 [monobasic], 136.9mM NaCl, 8.1mM Na3PO4 [dibasic heptahydrate] at pH 7.2). After embryo, cervix, ovaries, and adipose tissue were removed, uteri were diced and digested in an enzyme solution containing 300 µg/ml type II collagenase, 300 µg/ml type III trypsin, and 200 µg/ml deoxyribonuclease I. The digest was filtered through wire mesh with 1.5-mm openings and then through standard cheesecloth to remove large tissue clumps. The filtrate containing isolated cells was centrifuged to pellet the cells. After washing the cells repeatedly for two times with CMF-PBS, cells were seeded into flasks containing RPMI 1640 medium supplemented with 10% bovine calf serum (BCS). Cultured cells were incubated at 37°C in 5% CO2 atmospheric conditions. Medium was changed every 2 days, and cells were subcultured using crude trypsin to remove cells from flasks every 6–7 days, just prior to confluence. All cells were used at passage two. The smooth muscle character of the cultured cells was verified using indirect immunofluorescence labeling with mouse smooth muscle -actin antibodies as previously described (Loch-Caruso et al., 1990), and the purity of the cell cultures was 99–100%.
Microinjection.
Passage 1 cultured cells were removed from flasks by a 5-min exposure to 0.25% crude trypsin in CMF-PBS at 37°C and seeded into Corning polystyrene dishes at densities of 50,000 cells per dish. RPMI 1640 medium was supplemented with 10% BCS. Cells were incubated for 24 h at 37°C in a 5% CO2 atmosphere during which time cell attachment and growth occurred. Cells were microinjected with a mixed dye solution of 0.8% wt/vol Lucifer yellow CH and 0.02% propidium iodide. An injection pressure of 6.5 psi for 200 ms was used. Propidium iodide served as a marker of the injected cells by binding to nuclear DNA, and Lucifer yellow fluorescence in neighboring cells was used as a measure of gap junction intercellular communication. Lucifer yellow dye transfer from an injected cell to cells in direct contact with the injected cells by epifluorescence microscopy was scored and expressed as the percent dye transfer by dividing the number of adjacent cells that exhibited Lucifer yellow fluorescence by the total number of cells touching the injected cell, multiplied by 100. Cells were injected in RPMI 1640 medium over a 4-min period followed by rinsing with prewarmed CMF-PBS. Cells were scored in the order that they were injected. For experiments with antioxidants, cells were cotreated with -tocopherol, Def, or SOD in addition to 100µM 2,2'-DCB. Concentrations of inhibitors were selected according to previously published data.
Separation of cytosol and membrane fractions from uterine smooth muscle.
Uterine strips were homogenized with a Polytron homogenizer in ice-cold buffer (20mM Tris-HCl adjusted to pH 7.5, 250mM sucrose, and 3mM EGTA) containing 100 µl of protease inhibitor cocktails stock solution (1 tablet/1 ml H2O) (Roche, Indianapolis, IN). The homogenates were centrifuged at 100,000 x g for 60 min, and the supernatant was used as the cytosolic fraction. The resulting pellets were resuspended in homogenization buffer containing 1% Triton X-100, shaken at 4°C for 30 min, briefly sonicated, and centrifuged at 100,000 x g for 60 min. The resulting supernatant was used as the membrane fraction. The protein concentration of fractions was assayed by the Lowry method using a BioRad DC protein assay kit (BioRad, Hercules, CA) and bovine serum albumin as the standard.
Western blotting analysis for Cx43 phosphorylation.
Confluent cells in 75-cm2 plates were washed twice with cold CMF-PBS and lysed with 375 µl of cold lysis buffer (50mM Tris, 5mM EDTA, 150mM NaCl, 10mM NaF, pH 7.5) containing 100 µl of protease inhibitor cocktails stock solution (1 tablet/10 ml H2O) (Roche). Cells were harvested by scraping and collected in precooled microfuge tubes. The collected cells were treated with 550 µl of 40mM NaOH added to each microfuge tube, sonicated on ice for 5 s, and centrifuged at 16,600 x g for 30 min at 4°C. The pellets were resuspended in 100 µl of running buffer (62.5mM Tris, pH 6.8, 2% SDS, and 10% glycerol), sonicated for 5 s, and centrifuged at 16,600 x g for 5 min at 4°C. Protein concentration was determined by the BioRad DC protein kit (BioRad), and 20 µg of protein was loaded into the gel for each sample. Equal protein loading was verified by probing gels with anti-rabbit glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotech Inc, Santa Cruz, CA). Proteins were separated by SDS–polyacrylamide gel electrophoresis using 15% acrylamide. Following electrophoresis, proteins were transferred electrophoretically onto membranes and reacted for 1 h with rabbit polyclonal antibody for Cx43 phosphorylated at Ser255 (pCx43(S255)) (Santa Cruz Biotech Inc) at 1:100 dilution or rabbit polyclonal antibody for phosphorylated extracellular signal-regulated kinase (ERK) proteins (Santa Cruz Biotech Inc) at 1:100 dilution. The protein-primary complexes were probed with a 1:1000 dilution of alkaline phosphatase (AP)-conjugated anti-rabbit or anti-mouse antibodies (Amersham Life Sciences Products, Arlington Heights, IL), as appropriate, for 1 h.
Statistical analysis.
Data are reported as the mean ± SEM. Data analysis was conducted using SigmaStat (Jandel Scientific Software, San Rafael, CA). ROS measurement data, dye transfer data, and Western blot data were analyzed by one-way ANOVA. Contractility data were analyzed by two-way repeated measures ANOVA. Post hoc comparison of means was by Student-Newman-Keuls pairwise multiple comparison tests. A p value of 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Antioxidants on 2,2'-DCB–Induced Modification of Uterine Contractions
In order to investigate the role of oxidative stress in 2,2'-DCB–induced modification of uterine contraction, we measured the change of contractions in uterine strips treated with antioxidants after a 1-h exposure to 100µM 2,2'-DCB. After exposure to 2,2'-DCB, posttreatment with the antioxidants
-tocopherol (100µM), Def (50µM), or SOD (1000 U) significantly reversed 2,2'-DCB–induced decreases in amplitude and synchronization of uterine contractions (time and treatment effects, two-way repeated measures ANOVA, p 0.05) (Fig. 1). Compared with pretreatment (0-min exposure time), significant increases in contraction amplitude were observed within 1 h of antioxidant treatment and sustained for 3 h, whereas significant increases in contraction completion (synchronization) were observed at 2 and 3 h after initiation of antioxidant treatment (p 0.05). Unexpectedly, 0.001% corn oil, which was included as a vehicle control for -tocopherol, reversed 2,2'-DCB–induced decrease of contraction amplitude and synchronization to a similar extent as that observed with the antioxidants (p 0.05). Although the exact type of vegetable oil used by the manufacturer to prepare the -tocopherol is not known, the corn oil results indicate that the -tocopherol results may be confounded by vegetable oil vehicle. Regardless, the results with Def and SOD are consistent with a role for oxidative stress in 2,2'-DCB–induced modification of uterine contractions.
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Statistically different from 0 min within each treatment; (p Effect of 2,2'-DCB on Generation of Superoxide Anion in Uterine Tissue
In order to confirm that oxidative stress is induced in uterus by 2,2'-DCB, we examined whether 2,2'-DCB exposure of uterine tissue generates superoxide anion using the cytochrome c reduction assay, which detects extracellular superoxide anion. Uterine tissues treated with 100µM 2,2'-DCB showed an approximate fivefold increase of superoxide generation in 5 min (p
0.05; Fig. 2). However, there were no significant differences between treatment groups at 15, 30, and 60 min after 100µM 2,2'-DCB treatment.
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Effects of 2,2'-DCB on SOD Activity of Uterine Tissue
Because 2,2'-DCB induced superoxide anion generation in uterine tissue, we examined whether SOD activity was changed in uterine tissue by a 1-h exposure to 100µM 2,2'-DCB compared to uterine tissue that was untreated or was exposed for 1 h to dimethyl sulfoxide (solvent control). Uterine tissue exposed to 100µM 2,2'-DCB exhibited significantly decreased total SOD activity (p
0.05; Fig. 3), consistent with the increased generation of superoxide anion observed.
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Effect of 2,2'-DCB on Generation of ROS in Myometrial Cells
In order to examine whether 2,2'-DCB induces oxidative stress in myometrial cells, four biochemical assays that measure different end points were employed to assess responses in myometrial cell cultures exposed to 2,2'-DCB. When we measured superoxide generation using the cytochrome c reduction assay, there were no significant differences after 5, 15, 30, and 60 min of exposure to 50 or 100µM 2,2'-DCB compared with solvent controls, even though superoxide generation increased with time (Fig. 4). Treatment with 100µM pyrogallol was included as a positive control. Furthermore, myometrial cell cultures exposed to 30–100µM 2,2'-DCB did not increase reactive species production in the formazan assay and lipid peroxidation in the thiobarbituric acid reactive substances (TBARS) assay or show evidence of hydrogen peroxide formation in the DCF assay (data not shown).
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Effect of Antioxidants on 2,2'-DCB–Induced Inhibition of Gap Junctions
To investigate the role of oxidative stress on 2,2'-DCB–induced inhibition of myometrial gap junctions, myometrial cells in culture were treated for 1 h with 100µM 2,2'-DCB or cotreated with Def (50µM) or SOD (1000 U) and then assessed for Lucifer yellow dye transfer. The percentage of dye transfer decreased to 18% in cultures treated with 2,2'-DCB alone. Moreover, the percentage of dye transfer for cells cotreated with SOD or Def remained depressed compared with 2,2'-DCB only (Fig. 5). These results fail to support a role for oxidative stress in 2,2'-DCB–induced inhibition of myometrial gap junctions.
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Effect of Antioxidants on 2,2'-DCB–Induced Phosphorylation of Cx43
Previously, it was shown that 2,2'-DCB–induced inhibition of myometrial gap junctions is mediated through MAPK-induced phosphorylation of Cx43 at Ser255. In order to examine whether oxidative stress impacted on 2,2'-DCB–induced phosphorylation of Cx43 in myometrial cells, myometrial cells in culture were treated for 1 h with 100µM 2,2'-DCB alone or cotreated with 2,2'-DCB and either 50µM Def or 1000 U SOD. Proteins from treated cells were analyzed by Western blotting using an antibody against pCx43(S255) and normalizing band density to the housekeeping gene GAPDH. Compared with 2,2'-DCB–only, phosphorylated Cx43 protein was not changed by antioxidant cotreatment of myometrial cell cultures (Fig. 6).
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Effect of Antioxidants on 2,2'-DCB–Induced Phosphorylation of MAPK
The ERKs are the prototypic MAPKs and are activated via phosphorylation of tyrosine by MAPK/ERK kinase. In order to examine whether oxidative stresses have an impact on 2,2'-DCB–induced activation of ERK/MAPK in myometrial cells, Western blotting was performed for phosphorylated ERK. Western blot bands identified with phospho-ERK antibodies were normalized to GAPDH. The ratio of phospho-ERK to GAPDH was compared between cells that were exposed for 1 h to 100µM 2,2'-DCB or cotreated with 2,2'-DCB and either 50µM Def or 1000 U SOD. Densitometric analysis showed that antioxidant treatment did not change significantly the expression of the phospho-ERK compared to 2,2'-DCB only in myometrial cells (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oxidative stress has been implicated in various toxic effects and diseases by a growing scientific literature. Oxidative stress can be defined as a disturbance in the equilibrium status of prooxidant/antioxidant systems in intact cells. The antioxidant systems can be altered by inducing or repressing proteins that participate in these systems and by depleting cellular stores of antioxidant materials such as glutathione and vitamin E. Radical ROS such as superoxide anion and hydroxyl radical and nonradical ROS such as hydrogen peroxide disturb prooxidant/antioxidant systems and are correlated with damage in tissue and diseases (Fridovich, 1993
; Warner, 1994).
In contractility experiments to examine the role of oxidative stress in 2,2'-DCB–induced inhibition of uterine contraction, SOD was used as an antioxidant enzyme to catalyze superoxide anion to hydrogen peroxide (Woods, 2001). Hydrogen peroxide can be converted to water and oxygen molecule by catalase but, alternatively, may be converted to the highly reactive hydroxyl radical in a spontaneous reaction catalyzed by iron (Fenton reaction). The hydroxyl radical, product of the Fenton reaction, reacts with lipids and initiates the process of lipid peroxidation. Def is an iron scavenger and functions as an antioxidant under cellular conditions that deploy iron for the Fenton reaction. The reversal of 2,2'-DCB–induced inhibition of uterine contraction by SOD and Def suggests that oxidative stress is involved in 2,2'-DCB–induced inhibition of uterine contraction. This observation is consistent with the study of Horackova et al. (2000) that protective effects are observed with Def in H2O2-induced contractile arrest after irregular contractile activity.
In the present study, the effect of -tocopherol as an antioxidant was confounded by corn oil, which was included as a vehicle control treatment for the (unspecified) vegetable oil used by the manufacturer to dissolve -tocopherol. One possible explanation for the ability of corn oil to reverse 2,2'-DCB–induced modulation of uterine contraction is the presence of -tocopherol in the corn oil because -tocopherol is usually found in vegetable oils (corn, soybean, or cottonseed). Furthermore, corn oil contains unsaturated fatty acids that could react with hydroxyl radicals and thus may offer a scavenging effect. Another possible explanation is that corn oil modulates uterine contraction by an unknown mechanism that is independent of scavenging of ROS. There are few reports on the biological function of vegetable oils such as soybean oils and corn oils in the literature, especially related to the uterine contractions. A report that fennel essential oil reduces the intensity of oxytocin- and Prostaglandin E(2)-induced contractions (Ostad et al., 2001) provides no further insight into the reversal of 2,2'-DCB–induced inhibition of uterine contraction observed in the present study.
Previous reports showed that 2,2'-DCB generates ROS in various types of cells. Voie and Fonnum (2000) demonstrated the production of ROS by 2,2'-DCB in rat synaptosomes, and Voie et al. (1998) showed that 2,2'-DCB activates a respiratory burst and generates ROS in human granulocytes. In the present study, a surge of superoxide anion generation was observed in 2,2'-DCB–treated uterine tissue. In contrast, four different biochemical assays failed to detect evidence of 2,2'-DCB–induced oxidative stress in myometrial cells in culture. The contrast of results for measures of oxidative stress in cultured myometrial cells compared with uterine tissue suggests that ROS generation in the uterus may be localized in nonmyometrial cells. Alternatively, myometrial cells in culture may differ from myometrial cells in tissue with respect to characteristics that are important for 2,2'-DCB activation of oxidative stress mechanisms.
Superoxide anion was generated in uterine tissue after 5 min of exposure to 2,2'-DCB but returned to control levels after 15 min of exposure. Because superoxide anion has a very short half-life and the cytochrome c reduction assay depends on the reaction with extracellular superoxide anion, the cytochrome c reduction assay does not reflect cumulative superoxide anion generation at the later time points. This surge of superoxide anion after 5 min of exposure may function as an initiator for 2,2'-DCB–induced modification of uterine contractions by activation of signaling pathway sensitive to oxidative stress. Although our previous study showed mechanistic links between MAPK phosphorylation of Cx43, inhibition of myometrial gap junctions, and modulation of uterine contractions by 2,2'-DCB (Chung and Caruso, 2005), the present study indicates that 2,2'-DCB–induced oxidative stress is independent of inhibition of gap junctions, phosphorylation of Cx43, and activation of MAPK.
The endometrium may be a potential candidate site for superoxide anion generation in the uterus because ROS, including superoxide anion, are generated in the endometrium in the human uterus (Benedetto et al., 1981; Sugino et al., 1996). Superoxide anion is produced by a membrane-bound nicotinamide dinucleotide phosphate (NADPH) oxidase in phagocytes as well as in nonphagocytic cells, such as endothelial cells and vascular smooth muscle cells (Görlach et al., 2000). Furthermore, Sugino et al. (1996) concluded that SOD plays an important role in the accumulation of ROS by the findings such as increased ROS and lowered SOD activities in late–secretory phase endometrium. Further experiments to identify the source of superoxide anion generated by 2,2'-DCB, such as use of the NADPH oxidase inhibitor diphenyleneiodonium, will help to understand the mechanism by which oxidative stress plays a role in 2,2'-DCB–induced modification of uterine contractions. However, these experiments are beyond the scope of the present investigation.
The results of the present study are consistent with our previous report that 2,2'-DCB–induced decreases of amplitude and synchronization of uterine contractions are dependent on MAPK-induced phosphorylation of Cx43 and inhibition of myometrial gap junctions (Chung and Caruso, 2005). However, the data fail to corroborate oxidative stress as a mechanistic link between inhibition of myometrial gap junctions and modulation of uterine contractions by 2,2'-DCB. Although the uterine tissue results support a role for oxidative stress in 2,2'-DCB–induced modification of uterine contractions, experiments with the antioxidants SOD and Def in cultured myometrial cells suggest that inhibition of gap junction communication, activation of MAPK, and phosphorylation of Cx43 are independent of oxidative stress. Because of the inability of either SOD or Def to enter cells, the membrane-permeable ROS scavenger Tempol could be used in future experiments to strengthen conclusions about the lack of a role of ROS in the cultured myometrial cells. Nonetheless, SOD and Def were effective at reversing 2,2'-DCB–induced modification of uterine contractions. Moreover, the inability of SOD and Def to prevent 2,2'-DCB–induced phosphorylation of Cx43 at Ser255 and inhibition of gap junctions among rat myometrial cells in culture is in contrast to the preventative actions of the MAP2K1 inhibitor PD98059 (Chung and Caruso, 2005). Consequently, it is suggested that there are two different pathways for 2,2'-DCB–induced modification of uterine contractions. One pathway entails MAPK-dependent phosphorylation of Cx43 and inhibition of myometrial gap junctions, and the other pathway involves oxidative stress that is independent of myometrial gap junction inhibition.
Independence of 2,2'-DCB–induced oxidative stress from the 2,2'-DCB–induced MAPK signaling pathway suggests that oxidative stress may impact an alternative cell-signaling pathway. One such pathway could involve perturbation of calcium signaling. Goldhaber and Qayyum (2000) demonstrated that oxygen free radicals contribute to contractile failure mediated by decreased Ca2+ entry via L-type Ca2+ channels and reduced sarcoplasmic reticulum Ca2+ content. Blockage of calcium channels is a possible, though untested, mechanistic link between 2,2'-DCB–induced oxidative stress and modification of uterine contraction.
The present study shows that 2,2'-DCB modifies uterine contractions by an oxidative stress mechanism that is independent of a previously identified mechanism involving MAPK-dependent phosphorylation of Cx43 and inhibition of myometrial gap junctions. Additional studies to identify the source of oxidative stress and potential signaling pathways affected by oxidative stress in the uterus will help to understand the role of oxidative stress in 2,2'-DCB–induced modification of uterine contractions.
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NOTES |
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1 Present address: Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1683.
Disclaimer: Contents of the work are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
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ACKNOWLEDGMENTS |
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This study was supported by grant P42ES04911 from the National Institute of Environmental Health Sciences, National Institutes of Health, issued to R.L.C.
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