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Biology of Reproduction 64, 537-547 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Antioxidants Prevent {gamma}-Hexachlorocyclohexane-Induced Inhibition of Rat Myometrial Gap Junctions and Contractions1

Teresa R. Kriegera, and Rita Loch-Caruso2,a

a Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-2029

ABSTRACT

Lindane ({gamma}-hexachlorocyclohexane) is a commonly used pesticide that bioaccumulates in mammalian adipose tissue. Lindane inhibits gap junctional intercellular communication and oscillatory contractions of pregnant rat myometrium in vitro. The present study investigated the role of oxidative stress in lindane's inhibition of myometrial function in mid-gestation pregnant rat uteri. Lucifer yellow dye was microinjected into cultured myocytes to assess gap junctional intercellular communication. Lindane exposure (100 µM) resulted in a time-dependent, biphasic inhibition of dye transfer. This pattern of inhibition was also seen upon cell exposure to the pro-oxidant, tert-butyl hydroperoxide (100 µM). Lindane's initial and secondary-onset dye transfer inhibitions were reversed by cotreatment and pretreatment with the antioxidants, {alpha}-tocopherol (25–100 µM), diphenyl-1,4-phenylene diamine (10–30 µM), and superoxide dismutase (100–400 U/ml). D-mannitol (100–300 mM) also reversed lindane's initial dye transfer inhibition. Nitro blue tetrazolium reduction to formazan (measured spectrophotometrically) was elevated upon exposure of cultured cells to lindane or tert-butyl hydroperoxide, indicating the presence of reducing agents. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was also elevated in lindane-exposed cell cultures. {alpha}-Tocopherol reversed this elevation. Finally, uterine contractility was assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. Pretreatment with {alpha}-tocopherol prevented lindane's abolishment of uterine contractions in vitro. These data support the hypothesis that lindane inhibits uterine contractility and myometrial gap junctions by establishing an oxidative stress environment.

signal tranducers, uterus

INTRODUCTION

Lindane, the gamma isomer of hexachlorocyclohexane, is a commonly utilized pesticide that tends to bioaccumulate in human adipose tissue due to its lipophilic nature [1, 2]. Lindane toxicity commonly involves increased central nervous system excitability that can include muscle spasms and convulsions [3, 4]. Compared with studies of lindane's effects on the nervous system, relatively few studies have evaluated adverse effects on the reproductive system. Petrescu et al. [5] showed that lindane exposure led to increased gestation length in rats. Our laboratory has demonstrated decreased contractility of rat uterine strips exposed to lindane in vitro [6] as well as a lindane-induced inhibition of gap junctional intercellular communication in myometrial cell cultures [7]. Normal gap junctional intercellular communication between myometrial cells is believed to be necessary for proper coordination of smooth muscle contractions, allowing the generation of sufficient contractile force, which is essential for labor [8].

Normal cellular function depends on a balance between reactive oxygen species produced and antioxidant defense mechanisms available to the cell. Reactive oxygen species, including the superoxide radical, hydrogen peroxide, and the hydroxyl radical, arise as by-products of normal cellular metabolism or may be the consequence of exposure to certain toxicants [9, 10]. In instances in which the existing enzymatic and nonenzymatic cellular antioxidants are unable to counteract the reactive oxygen species, a condition known as oxidative stress occurs [11]. In such cases of overwhelmed antioxidant defense, cellular function can be affected and cells may be damaged [11, 12]. The present study evaluated the mechanism by which lindane inhibits rat myometrial function and hypothesized that lindane's inhibition is subsequent to the induction of an oxidative stress.

MATERIALS AND METHODS

Chemicals

Lindane ({gamma}-hexachlorocyclohexane, 99% purity) was purchased from Sigma Chemical Company (St. Louis, MO). Lucifer yellow and propidium iodide dyes were obtained from Molecular Probes (Eugene, OR). RPMI cell culture medium was purchased from Gibco (Grand Island, NY) and was supplemented with iron and transferrin-enriched bovine calf serum (BCS; Hyclone, Logan, UT). N,N'-diphenyl-1,4-phenylene diamine (DPPD) was purchased from Aldrich Chemical Company (Milwaukee, WI). Nitro blue tetrazolium (NBT), Cu/Zn-superoxide dismutase (SOD), (±)-{alpha}-tocopherol (vitamin E), tert-butyl hydroperoxide (tBH), and all other chemicals used were obtained from Sigma.

Animals

Time-pregnant Sprague-Dawley rats were obtained from the breeding colony of the University of Michigan's Reproductive Sciences Program (Ann Arbor, MI). The rats were between 60 and 90 days of age and weighed between 180 and 220 g. The animals were housed at ambient temperature (24 ± 1°C) under a 12-h light schedule. The day of detection of spermatozoa in the vaginal smear was designated as Day 0 of pregnancy. The rats were cared for and handled in accordance with the Guide for Care and Use of Laboratory Animals (published by the National Academy of Science, 1996) and protocols approved by the University Committee on Use and Care of Animals of the University of Michigan.

Cell Isolation and Culture

Myometrial smooth muscle cells were isolated from mid-gestation (Day 10) Sprague-Dawley rats that had been anesthetized with ether and killed by exsanguination or cardiac puncture. Upon removal, uteri were immediately placed in ice cold physiologic saline solution (PSS) containing 135 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1 mM CaCl2, 1 mM Hepes, and 5 mM glucose, adjusted to pH 7.4 with 1 N NaOH. Myometrial cells were isolated as previously described by Caruso et al. [13]. Briefly, after embryos, cervix, ovaries, and adipose tissue were removed, uteri were diced and digested in an enzyme solution containing type II collagenase, type III trypsin, and deoxyribonuclease I (300, 300, and 200 µg/ml, respectively). The digest was filtered through wire mesh with 1.5-mm openings, and then through standard cheesecloth to remove large tissue clumps, and the filtrate containing isolated cells was centrifuged to pellet the cells. After repeated washing of the cells with calcium/magnesium-free PBS (CMF-PBS; 2.68 mM KCl, 1.5 mM K3PO4 [monobasic], 136.9 mM NaCl, 8.1 mM Na3PO4 [dibasic heptahydrate] at pH 7.2), cells were seeded into flasks containing RPMI medium supplemented with 10% BCS. Cultured cells were incubated at 37°C with 5% CO2 atmospheric conditions. Medium was changed every 2 days and cells were subcultured every 6–7 days, just prior to confluence. Unless otherwise indicated, all experiments utilized cultured myometrial cells at passage 2. Isolated cells were characterized as smooth muscle cells by indirect immunofluorescence labeling with mouse anti-{alpha}-smooth muscle-specific actin monoclonal antibody as described previously by Caruso et al. [13]. Alpha-actin labeling indicated that cultures were at least 99% smooth muscle.

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 were seeded into 35-mm and 60-mm Corning polystyrene dishes at densities of 40 000 and 60 000 cells per dish, respectively. RPMI medium supplemented with 10% BCS was added to the dishes at 1.5 ml for 35-mm dishes or 3 ml for 60-mm dishes. Cells were then incubated for 24–36 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 1% w/v Lucifer yellow CH and 0.1% propidium iodide in CMF-PBS. Propidium iodide served as a marker of the injected cells by binding to nuclear DNA and Lucifer yellow fluorescence in neighboring cells was used to assess gap junctional intercellular communication. An injection pressure of 6.5 psi for 200 msec was used. Lucifer yellow dye transfer from the injected cell to cells in direct contact with the injected cell was scored and expressed as the number of adjacent cells that exhibited Lucifer yellow fluorescence divided by the total number of cells touching the injected cell, multiplied by 100%. Cells were injected in RPMI medium over a 4-min period followed by rinsing with prewarmed CMF-PBS. Cells were scored 1 min later in warm CMF-PBS. The scoring period was restricted to 4 min and cells were scored in the order that they were injected. RPMI medium used throughout all microinjection experiments was supplemented with 10% BCS.

To replicate lindane's inhibitory effect on dye transfer as previously reported by our laboratory [7], plated cells were incubated in RPMI medium containing 100 µM lindane (derived from a stock solution of 50 mM lindane in dimethyl sulfoxide [DMSO]) for 1 h at 37°C in a 5% CO2 atmosphere. Cells were then microinjected in the lindane-containing medium for the zero-hour time point or, alternatively, were rinsed three times with prewarmed CMF-PBS and incubated with fresh RPMI medium (without lindane) at 37°C in a 5% CO2 atmosphere for 0.25, 0.5, 1, 4, or 24 h after rinsing. These cells were then microinjected in the lindane-free RPMI medium. Individual culture dishes were utilized for one time point only, subsequent to random assignment of cell-containing dishes to the various treatment groups. Solvent controls were incubated for 1 h in RPMI medium containing 0.2% DMSO (the solvent concentration used to deliver lindane) and treated in a manner identical to the cultures exposed to lindane.

The effects of distinct antioxidants on lindane-induced dye transfer inhibition were evaluated. For {alpha}-tocopherol, cells were incubated for 1 h at 37°C in RPMI medium containing either 100 µM lindane alone or 25, 50, or 100 µM (±) {alpha}-tocopherol (derived from a stock solution of 100 mM {alpha}-tocopherol in DMSO) in addition to 100 µM lindane. Treated cells were microinjected in chemical-containing RPMI medium. Additional plates of cells were pretreated for 15 min with 10, 20, or 30 µM DPPD before addition of 100 µM lindane, were cotreated with 100 µM lindane and 100, 300, or 400 U/ml SOD, or were cotreated with 100 µM lindane, and 100, 200, or 300 mM D-mannitol. Cells were microinjected with Lucifer yellow dye immediately at the end of a 1-h incubation in chemical-containing RPMI medium. With three of the antioxidants, a single concentration was used to evaluate the 24-h effect on lindane-induced inhibition of dye transfer. Using the same initial exposure protocol, cells were cotreated or pretreated with 100 µM {alpha}-tocopherol, 30 µM DPPD, or 100 U/ml SOD in addition to 100 µM lindane in RPMI medium. Following a 1-h incubation at 37°C, cells were rinsed three times with prewarmed CMF-PBS. Cells were then incubated an additional 24 h at 37°C in fresh RPMI medium (containing no chemicals) and were microinjected at the end of the 24 h in chemical-free RPMI medium.

Additional experiments evaluated the effects of the oxidative stress inducer, tBH [14, 15]. Cells were incubated at 37°C for 30 min in RPMI medium (supplemented throughout the experiments with 10% BCS) containing 100 µM tBH. At the end of this incubation time, cells were either assessed for dye transfer immediately or were rinsed three times with warm CMF-PBS and then incubated with fresh RPMI medium for additional periods of 0.25, 0.5, 1, or 24 h prior to microinjection. Individual culture dishes were utilized for one time point only, subsequent to random assignment to the various treatment groups. Corresponding controls were incubated for 30 min in chemical-free RPMI medium and then rinsed three times with warm CMF-PBS and handled in an identical manner as cultures exposed to tBH. Separate experiments evaluated dye transfer as a function of tBH exposure length. Cells were incubated at 37°C in RPMI medium (supplemented throughout the experiments with 10% BCS) containing 100 µM tBH. Cells were microinjected in the tBH-containing medium at 10, 20, 30, and 60 min of tBH exposure. Individual culture dishes were utilized for one time point only, following random assignment to the various treatment groups. Corresponding controls were incubated in chemical-free RPMI medium and handled in an identical manner as cultures exposed to tBH.

Residue Analysis

Cultured myometrial cells were grown to confluency in 175-mm flasks. Cells were then incubated for 1 h in RPMI medium (supplemented with 10% BCS) containing 100 µM lindane (derived from a stock solution of 50 mM lindane in DMSO) at 37°C in a 5% CO2 atmosphere. At the end of the 1-h incubation, the lindane-containing medium was decanted and cells were either rinsed three times with prewarmed CMF-PBS or were immediately processed without rinsing. In each case, cells were removed from flasks with a 5-min exposure to 0.25% crude trypsin in CMF-PBS at 37°C. Cells were then pelleted by a 5-min centrifugation at 125 x g at 4°C. Subsequently, cells were resuspended in 1 ml CMF-PBS and frozen and stored at 0°C. Small aliquots of each sample were withdrawn prior to freezing to determine total cell numbers. Samples were analyzed for lindane content by the National Institute of Environmental Health Sciences (NIEHS) Analytical Core at Michigan State University, East Lansing, Michigan. A Perkin-Elmer Autosystem (Norwalk, CT) gas chromatograph with a dual column electron-capture configuration was used to quantify and confirm the identity of the analyte. Lindane levels were expressed as parts per billion (ppb) lindane per 1000 cells.

Formazan Assay

Cultured myometrial cells were plated at passage 1 into 60-mm dishes at a density of 80 000 cells per dish using crude trypsin to remove cells from flasks as described for the microinjection procedure. Freshly plated cells were incubated in RPMI medium (with 10% BCS) for 48 h at 37°C in a 5% CO2 atmosphere to allow cell attachment and growth to approximately 75% confluence. A modification of the formazan assay described by Mochida et al. [16], based upon that of Rook et al. [17], was utilized. Briefly, nitro blue tetrazolium was dissolved at 2 mg/ml in 37°C PBS (0.9 mM CaCl2, 2.68 mM KCl, 1.47 mM K3PO4 [monobasic], 0.5 mM MgCl2 [hexahydrate], and 8 mM Na3PO4 [dibasic heptahydrate] at pH 7.2). Lindane was added at 50, 100, or 200 µM final concentration to the NBT solution. {alpha}-Tocopherol was added to separate NBT solution at final concentration of 100 µM. After rinsing once with prewarmed CMF-PBS, cells were incubated in chemical-containing NBT solution for 45 min at 37°C in a dark, 5% CO2 environment. At the end of 45 min, the NBT solution was decanted and the cells were rinsed and fixed by washing three times with 70% methanol. Cells were allowed to air dry and were then disrupted with 93 µl of 2 M KOH. Upon addition of 110 µl DMSO to solubilize formazan deposits, formazan content was measured spectrophotometrically at 630 nm.

Lipid Peroxidation Quantification

Lipid peroxidation was evaluated by the thiobarbituric acid method based upon the assay described by Agostinho et al. [18]. Briefly, passage 2 cultured myometrial cells were removed from culture flasks with a 5-min exposure to 0.25% crude trypsin in CMF-PBS at 37°C. Dissociated cells were rinsed of trypsin with the addition of trypsin-free CMF-PBS followed by a 4-min centrifugation at 125 x g and 4°C. The supernatant was decanted, cells were resuspended in fresh CMF-PBS, and recentrifuged for 4 min to complete trypsin removal. After decanting the supernatant from the pellets a second time, cells were suspended in PBS and aliquoted into individual microfuge tubes so that each contained 50 000 cells suspended in 0.5 ml PBS and either 100 µM lindane, 100 µM {alpha}-tocopherol, 100 µM lindane plus 100 µM {alpha}-tocopherol, or 0.2% DMSO (solvent control). Cells were incubated in the chemical-containing medium for 1 h at 37°C. Subsequently, cells were pelleted by centrifugation and the supernatant was decanted. To each cell pellet, 250 µl thiobarbituric acid assay solution (15% trichloroacetic acid, 0.75% thiobarbituric acid, 0.25 M HCl, and 0.015% butylated hydroxytoluene [BHT]) was added and samples were incubated for 30 min at 100°C. At the end of the incubation period, samples were centrifuged at 95.5 x gav for 10 min. Lipid peroxidation was quantified by reading the absorbence of each supernatant at 530 nm and calculating the amount of thiobarbituric acid-reactive substances (TBARS) formed using a molar extinction coefficient of 1.56 x 105 M-1·cm-1.

Uterine Contractility

Measurements of spontaneous oscillatory contractions were based on the procedure described by Tsai et al. [19]. Briefly, 1-mm-wide by 20-mm-long uterine strips were excised from gestation Day 10 rats, pooled, and randomly hung in standard muscle baths filled with 37°C physiological saline solution (PSS; 116 mM NaCl, 21.9 mM NaHCO3, 11.1 mM dextrose, 4.6 mM KCl, 1.16 mM MgSO4 [7 H2O], 1.16 mM NaH2PO4 [H2O], 1.8 mM CaCl2 [2H2O], and 2.6 mM EDTA at pH 7.4). Upon establishment of regular oscillatory contractions, detected by isometric force transducers, individual strips were challenged with a depolarizing concentration of 60 mM KCl and the resulting contraction measured to obtain the maximal contractile force of that strip. Treatments were randomized to the five muscle baths available and each bath contained two uterine strips. In the first experiment, uterine strips were exposed to 50 µM lindane or 0.1% DMSO (solvent control), with or without a 15-min pretreatment with 100 µM {alpha}-tocopherol. In other experiments, tBH was added to individual baths at final concentrations of 50 or 100 µM, and control strips were exposed to solvent (distilled, deionized water [DDW], 0.14% final concentration). Contraction amplitudes and frequencies were measured and averaged over 10-min intervals. The mean amplitude and frequency of the 10-min interval just prior to addition of test chemical was used as the basal amplitude and frequency, respectively, for individual strips. The response to treatment for each strip was expressed as percent basal oscillatory activity as follows:

Tissue viability was ensured by rechallenging strips with 60 mM KCl after the final time interval.

Statistical Analysis

Unless otherwise indicated, data are reported as the mean ± SEM. Data analysis was conducted using SigmaStat (Jandel Scientific Software, San Rafael, CA). Lindane and tBH dye transfer data over time were analyzed by two-way ANOVA. Contractility data were analyzed by two-way repeated measures ANOVA. All other data were analyzed by one-way ANOVA. Post-hoc comparison of means were by Student-Newman-Keuls pairwise multiple comparison tests. A P value of <= 0.05 was considered significant.

RESULTS

Intercellular transfer of Lucifer yellow dye (injected into individual cultured cells) was used to monitor lindane's effects on gap junctional intercellular communication. Lindane inhibited dye transfer among cultured rat myometrial cells in a time-dependent, biphasic manner, whereas treatment with DMSO alone (solvent control) permitted high levels of dye transfer (>97%) at all time points (Fig. 1A). A 1-h exposure to 100 µM lindane inhibited dye transfer to 2.61% of adjoining cells compared with 97.9% of adjoining cells in solvent controls (Fig. 1A, 0 h time point). This phenomenon will be referred to as lindane's acute inhibition of dye transfer. Removal of the lindane-containing medium initially resulted in rapid reversal of the dye transfer inhibition. Dye transferred to 56.6% of adjoining cells 0.25 h after rinsing and returned to control levels (98.1%) by 0.5 h after rinsing lindane-exposed cells (Fig. 1A). Although control levels of dye transfer (91.5%) were also observed 1 h after rinsing of lindane-exposed cultures, a secondary, delayed-onset inhibition developed with dye transfer in lindane-exposed cultures inhibited to 55.2%, 57.3%, and 57.6% at 2, 4, and 24 h, respectively, following lindane removal (Fig. 1A, P < 0.05 compared with time-matched controls). This secondary inhibition observed subsequent to lindane rinsing will be referred to as lindane's delayed inhibition of dye transfer. The rinsing protocol utilized throughout the experiments effectively removed lindane from our cells as evidenced by residue analysis data. Lindane levels averaged 379.2 ± 16.7 ppb lindane/1000 cells in nonrinsed myometrial cultures versus only 0.878 ppb lindane/1000 cells in rinsed cultures (n = 3 for nonrinsed cells, n = 2 for rinsed cells).



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  		FIG. 1. Time-dependent inhibition of Lucifer yellow dye transfer in myometrial cell cultures before rinsing (0 h) and after rinsing (0.25–24 h) to remove exposure medium. A) Exposure to 100 µM lindane for 1 h; there were 6–8 dishes per group with a minimum of six cells microinjected with dye per dish. B) Exposure to 100 µM tBH for 30 min; there were 3–4 dishes per group with at least 10 cells microinjected with dye per dish. Bars represent the means ± SEM. Means that are significantly different from each other (P < 0.05) are labeled with different letters

Similarly, a 100 µM concentration of tBH was utilized to assess effects of a known inducer of oxidative stress on Lucifer yellow dye transfer among cultured myometrial cells. A 30-min exposure to 100 µM tBH reduced dye transfer to 4.79% of adjoining cells compared with 97.9% of adjoining cells in solvent controls (Fig. 1B, 0 h time point). Removal of the tBH-containing medium resulted in rapid reversal of the dye transfer inhibition (Fig. 1B). By 0.25 h after rinsing, dye transfer in tBH-exposed cultures was partially reversed to 56.7%, compared with 98.9% dye transfer in time-matched controls. Compared with corresponding controls, full reversal was seen by 0.5 h after rinsing in tBH-exposed cultures (90.0% dye transfer), and dye transfer remained similar to control levels at 1 h following tBH rinsing (96.7%). This paralleled the pattern of dye transfer inhibition seen following exposure of cells to lindane (Fig. 1A). Furthermore, as with lindane, a secondary, delayed-onset inhibition developed following tBH exposure; dye transfer was inhibited to 26.4% at 24 h following tBH removal compared with time-matched control dye transfer of 99.2% (Fig. 1B, P < 0.05).

The hypothesis that lindane-induced inhibition of myometrial gap junctions is subsequent to oxidative stress induction was further examined by studying the ability of lindane to stimulate generation of reducing agents by measuring NBT dye conversion to formazan [16, 17]. A 45-min exposure to 50, 100, or 200 µM lindane increased formazan deposition 180%, 272%, and 456% relative to controls, respectively (Fig. 2, P < 0.05). Cotreatment with the antioxidant, {alpha}-tocopherol (100 µM), reversed the formazan increase seen with 100 µM lindane alone (Fig. 2, P < 0.05). The increased formazan deposition in lindane-exposed cultures was clearly evident by microscopic examination prior to cell lysis and formazan solubilization (Fig. 3).



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  		FIG. 2. Formazan deposition in cultured myometrial cells preloaded with NBT and exposed to 0.2% DMSO (solvent control), lindane (50, 100, or 200 µM), or 100 µM lindane + 100 µM {alpha}-tocopherol. Bars represent the means ± SEM of 6–8 dishes per treatment, expressed as percent control. Means that are significantly different from each other (P < 0.05) are labeled with different letters



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  		FIG. 3. Micrograph showing formazan deposition in cultured myometrial cells prior to cell disruption and formazan solubilization. A) Control cells exposed to 0.2% DMSO for 45 min. B) Lindane-treated cells exposed to 100 µM lindane for 45 min. The arrow shows a representative area of blue formazan deposits within the lindane-treated cells

Because {alpha}-tocopherol reversed lindane-induced formazan deposition, subsequent experiments evaluated effects of {alpha}-tocopherol and other antioxidants on lindane-induced dye transfer. In each case, treatment with antioxidants reversed lindane's acute inhibition of dye transfer. Treatment with 100 µM lindane for 1 h inhibited dye transfer to 2.61% (Fig. 4, A–C). {alpha}-Tocopherol (vitamin E) reversed lindane's inhibitory effect on dye transfer in a concentration-dependent manner (Fig. 4A). Cotreatment with 25, 50, or 100 µM {alpha}-tocopherol allowed dye transfer levels of 8.54%, 42.8%, and 95.8%, respectively, in the presence of 100 µM lindane. {alpha}-Tocopherol alone, at the highest concentration utilized, had no effect on dye transfer compared with controls. Similarly, pretreatment with 10, 20, or 30 µM DPPD reversed lindane's effect in a concentration-dependent manner, with dye transfers of 59.3%, 91.6%, and 97%, respectively, compared with 2.61% in cultures treated with lindane alone (100 µM for 1 h; Fig. 4B). The concentration-dependent effects of SOD are shown in Figure 4C. Cotreatment with 100, 300, or 400 U/ml SOD increased dye transfer to 31.6%, 67.8%, and 99.2%, respectively, in the presence of 100 µM lindane compared with 2.61% in cultures treated with lindane alone (Fig. 4C). Neither SOD nor NPPD, at the highest concentrations used, had significant effects on dye transfer compared with solvent controls.



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  		FIG. 4. Concentration-dependent reversal of lindane's acute Lucifer yellow dye transfer inhibition in myometrial cells by antioxidants. Cultured myometrial cells were exposed to 100 µM lindane for 1 h with A) {alpha}-tocopherol cotreatment, B) DPPD pretreatment, or C) SOD cotreatment. Each bar represents the mean ± SEM of at least six dishes per group with a minimum of six cells microinjected with dye per dish. Different letters indicate significantly different mean values (P < 0.05). Error bars not visible are too small for graphical display

In addition to reversing lindane's initial, acute inhibition of dye transfer, antioxidants blocked the secondary, delayed-onset inhibition of dye transfer 24 h after removal of lindane exposure medium (Fig. 5). When cells were treated for 1 h with either 100 µM {alpha}-tocopherol, 30 µM DPPD, or 100 U/ml SOD, concurrent with 100 µM lindane, dye transfers were 96.3%, 98.6%, and 97.1%, respectively, 24 h after removal of the exposure medium. Control dye transfer levels averaged 97.7% and lindane treatment alone inhibited dye transfer to 52.8% 24 h after a 1-h exposure to solvent or 100 µM lindane (Fig. 5, P < 0.05). Thus, each antioxidant fully reversed lindane's secondary delayed-onset inhibition of dye transfer in addition to lindane's acute inhibition of dye transfer.



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  		FIG. 5. Reversal of lindane's secondary, delayed-onset inhibition of Lucifer yellow dye transfer by antioxidants, assessed 24 h after removal of exposure medium. Cells were exposed for 1 h to 0.2% DMSO (control), 100 µM lindane, 100 µM {alpha}-tocopherol, 100 U/ml SOD, or 30 µM DPPD. Other cultures were cotreated for 1 h with 100 µM lindane and 100 µM {alpha}-tocopherol, 100 U/ml SOD, or 30 µM DPPD (after a 15-min DPPD pretreatment). Bars represent the means ± SEM of 6–8 dishes per groups with a minimum of six cells microinjected with dye per dish. The asterisk indicates that the value is significantly different from control (P < 0.05)

Additional experiments further evaluated the presence and role of distinct reactive oxygen species. As seen previously, a 1-h exposure to 100 µM lindane alone diminished dye transfer to 4.57% compared with 96.3% for solvent controls (0.2% DMSO; Fig. 6, P < 0.05). D-mannitol, a specific inhibitor of the hydroxyl radical [20–22], reversed lindane's acute inhibitory effect on dye transfer in a concentration-dependent manner (Fig. 6). Dye transfers between cultured cells cotreated with 100, 200, or 300 mM D-mannitol in addition to 100 µM lindane were 26.5%, 71.1%, and 96.4%, respectively. A 1-h exposure to 300 mM D-mannitol alone had no significant effect on dye transfer (96.7% dye transfer observed) compared with controls.



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  		FIG. 6. Concentration-dependent reversal of lindane's acute inhibition of Lucifer yellow dye transfer by D-mannitol cotreatment. Bars represent the mean percent dye transfers ± SEM of five dishes per group with a minimum of six cells microinjected with dye per dish. Different letters indicate significantly different mean values (P < 0.05)

To further assess the presence and consequences of hydroxyl radical, an experiment evaluated lipid peroxidation in cultured cells exposed to lindane. TBARS were used as a measure of the amount of lipid peroxidation present. A 1-h incubation of cultured cells with 100 µM lindane resulted in an increase of TBARS to 289% of control levels (Fig. 7, P < 0.05). Cotreatment with 100 µM {alpha}-tocopherol prevented the increase in TBARS that resulted from 100 µM lindane alone. Furthermore, 100 µM {alpha}-tocopherol by itself reduced TBARS to 85.9% of control amounts.



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  		FIG. 7. TBARS in cultured myometrial cells exposed to 0.2% DMSO (solvent control), 100 µM lindane, 100 µM {alpha}-tocopherol or 100 µM {alpha}-tocopherol plus 100 µM lindane. Values are expressed as percent control and represent the mean ± SEM levels of 3–5 culture dishes per treatment group. Different letters indicate significantly different mean values (P < 0.05)

For further assessment of effects of hydrogen peroxide on rat myometrium, the model pro-oxidant, tBH, was again utilized and, in this case, cell cultures were exposed to the chemical for various lengths of time and tBH medium was not removed from cultures prior to microinjection. As previously observed in this study, tBH dramatically inhibited Lucifer yellow dye transfer between cultured myometrial cells, suggesting impairment of gap junctional intercellular communication. Cells exposed to 100 µM tBH for 10, 20, 30, or 60 min demonstrated time-dependent depressed dye transfers of 38.1%, 26.2%, 4.79%, and 0.63%, respectively, compared with control dye transfers of 97.9%, 97.1%, 97.9%, and 99.2%, respectively (Fig. 8, P < 0.05).



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  		FIG. 8. Time course of inhibition of Lucifer yellow dye transfer in cultured myometrial cells continuously exposed to 100 µM tBH. Each bar represents the mean percent dye transfer ± SEM of 6–8 dishes per group with a minimum of six cells microinjected with dye per dish. Different letters indicate significantly different mean values (P < 0.05)

Because inhibition of gap junctional intercellular communication can negatively affect the generation of coordinated, functional uterine contractions [8], the effects of tBH on uterine contractility were also evaluated. Whereas uterine contractions of control strips remained unchanged over time with exposure to the solvent DDW, the oscillatory activity of uterine strips exposed to 50 or 100 µM tBH hydroperoxide decreased in time- and concentration-dependent manners (Fig. 9). Exposure to tBH did not significantly depress oscillatory activity with short exposure durations. After 40 min, however, 50 µM tBH depressed oscillatory activity to 24.2% and 16.1% basal oscillatory activity for the time intervals 40–50 min and 50–60 min, respectively (P < 0.05 compared with time-matched controls and with shorter duration intervals). Exposure to 100 µM tBH produced a more rapid and greater reduction of oscillatory activity: significant reductions of oscillatory activity to 22.7% and 6.80% basal oscillatory activity were observed during the 30- to 40-min and 40- to 50-min exposure intervals, respectively (P < 0.05 compared with time-matched controls and shorter exposure duration intervals). Contractions were abolished in all strips exposed to 100 µM tBH for 50 min. As shown in the polygraph tracing in Figure 10, 100 µM tBH slightly depressed the amplitude and frequency of contraction before oscillations ceased.



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  		FIG. 9. Time- and concentration-dependent inhibition of oscillatory activity by tBH, defined as the product of mean contractile amplitude and frequency (see Materials and Methods). Each bar represents the mean oscillatory activity (expressed as percent basal activity) ± SEM over 10-min intervals of tBH or solvent control (0.14% DDW) exposure. n = 3–8 uterine strips per treatment group. Different letters indicate mean values are significantly different (P < 0.05). Data and error bars not visible are too small to be depicted graphically



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  		FIG. 10. Representative tracings of the contraction patterns of uterine strips exposed to 100 µM tBH (top tracing) or to 0.14% DDW (solvent control; bottom tracing). Arrowheads mark time of tBH or DDW addition. Arrow marks time of 60 mM KCl addition at the end of the experiment

Similar to tBH, lindane inhibited oscillatory activity of rat uterine strips hung in standard muscle baths (Fig. 11, A and B). Although oscillatory activity of control uterine strips (0.1% DMSO, final concentration) remained near 100% basal levels throughout the duration of the experiment (data not shown), 50 µM lindane decreased uterine contractions in a time-dependent manner (Fig. 11A, without {alpha}-tocopherol pretreatment). The lower part of Figure 11B shows a representative polygraph tracing demonstrating lindane's inhibition of uterine contractions over time. During the first 10-min interval following the addition of 50 µM lindane (final concentration) in bolus form, mean oscillatory activity remained comparable to control activity (98.3% basal level). However, by 10–20 min following lindane addition, mean oscillatory activity fell to 28.3% basal level and by 20–30 min after lindane addition, mean oscillatory activity was further diminished to 5.33% of basal levels (Fig. 11A, P < 0.05 compared with shorter exposure durations). Lindane completely abolished uterine contractions of all strips by 30 min (Fig. 11A). A 15-min pretreatment with 100 µM {alpha}-tocopherol prevented the inhibition of uterine contractility caused by 50 µM lindane (Fig. 11A, P < 0.05 for exposure durations exceeding 10 min). During the first 10 min following lindane addition, strips pretreated with {alpha}-tocopherol averaged an oscillatory activity of 106.5% basal levels. By 10–20 min after lindane addition, oscillatory activity in these strips was 95.3% of basal amounts and remained at 95% basal levels during the 20-to 30-min exposure interval. While contractions were completely abolished during the 30- to 40-min interval following lindane addition in strips exposed to lindane alone, strips pretreated with {alpha}-tocopherol maintained a mean oscillatory activity of 83.2% of basal level during the same time interval after lindane addition. A representative polygraph tracing showing the protective effect of {alpha}-tocopherol against lindane's abolishment of uterine contractions is shown in Figure 11B.



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  		FIG. 11. {alpha}-Tocopherol protection against lindane-induced inhibition of uterine contractility. Uterine strips were pretreated with 100 µM {alpha}-tocopherol or with 0.2% DMSO without vitamin E pretreatment (solvent control) for 15 min prior to the addition of 50 µM lindane, and contractions of uterine strips were followed over 40 min. A) Oscillatory activity, defined as the product of mean contractile amplitude and frequency (see Materials and Methods) following lindane addition. Time intervals depicted are 10-min increments after lindane addition to the muscle baths. Values represent the means ± SEM, expressed as percent basal oscillatory activity. n = 6–8 uterine strips. Different letters indicate mean values are significantly different. Data and error bars not visible are too small to be depicted graphically. B) Representative tracings of the contraction patterns of lindane-exposed uterine strips with (top tracing) and without (bottom tracing) {alpha}-tocopherol pretreatment. Arrowheads mark time of 50 µM lindane addition. Arrow marks time of 60 mM KCl addition at the end of the experiment. Pretreatment amplitude differences between the top and bottom strip tracings reflect individual variation seen among uterine strips, and this variation is adjusted for by expressing data as percent basal oscillatory activity

DISCUSSION

Lindane creates a pro-oxidant environment in rat myometrial cells, evidenced by increased formazan deposition in cells preloaded with NBT. This yellow dye is converted to insoluble blue formazan in the presence of reducing agents such as the superoxide anion, and the formazan assay has been utilized in various cell types as an indicator of oxidative stress [16, 17]. Lindane induction of oxidative stress has been demonstrated in other tissue types [23–25] including reproductive tissue [26, 27]. However, this study is the first to provide evidence of lindane-induced oxidative stress in myometrium. The lindane-induced elevation of NBT conversion to formazan could be due to an elevation in amounts of reactive oxygen species produced or to depressed functioning of antioxidant systems, the net result, in either case, being an oxidative stress condition.

Lindane's inhibitory effects on myometrial functions, observed previously by our laboratory [6, 7], were replicated in this study. Exposure of cultured rat myometrial cells to lindane resulted in a near-abolishment of cell-to-cell Lucifer yellow dye transfer, an indicator of gap junctional intercellular communication. Lindane-induced inhibition of gap junctions has been reported in various cell types [28–31] including those derived from reproductive tissue [32]. Gap junctional intercellular communication provides a mechanism by which adjacent myometrial cells exchange intercellular signals leading to the synchronous, coordinated contractions necessary for labor [8]. Thus, adverse effects of lindane on myometrial gap junctional intercellular communication in vivo would be expected to negatively affect parturition via toxicant-related uterine atony.

Although removal of lindane-containing medium initially reversed inhibition of gap junctional intercellular communication, a secondary inhibition developed following removal of the exposure medium. Because residue analysis suggested near complete removal of lindane following the rinsing protocols conducted in this study, it is unlikely that this delayed-onset inhibition was due to continued significant presence of the toxicant in its parent form. Rather, it is possible that the initial lindane pulse commences a secondary process leading to the secondary, delayed-onset gap junctional intercellular communication inhibition.

Reactive oxygen species possess properties that enable their participation as secondary messengers [33, 34] and could act by altering function, formation, processing, or turnover of gap junction proteins. Indeed, reactive oxygen species-mediated inhibition of gap junctional intercellular communication has been demonstrated in other cell types [29, 35, 36]. Tert-butyl hydroperoxide is a known inducer of oxidative stress in vitro [14, 15]. Exposure of myometrial cells to tBH resulted in a biphasic pattern of inhibition of gap junctional intercellular communication nearly identical to that of lindane. Therefore, the extent and the manner of inhibition observed upon lindane exposure paralleled those observed under oxidative stress conditions induced by a known pro-oxidant.

Further support that lindane induces an oxidative stress condition in the cultured cells was provided by experiments with antioxidants. The nonenzymatic antioxidants, {alpha}-tocopherol and DPPD, reversed lindane's acute inhibition of Lucifer yellow dye transfer in a concentration-dependent manner. Furthermore, both antioxidants prevented lindane's secondary, delayed-onset inhibition of gap junctional intercellular communication. Addition of the enzymatic antioxidant, SOD, which is normally present in cellular cytoplasm [37], similarly reversed lindane's acute and delayed inhibitory actions on Lucifer yellow dye transfer. These results suggest that the pathways leading to the biphasic inhibition of myometrial gap junctional intercellular communication caused by lindane involve increased production of reactive oxygen species and/or adverse effects on normal cellular antioxidant function. Further studies quantifying reactive oxygen species levels and total antioxidant capability in the presence of lindane, with and without antioxidant cotreatment, would confirm lindane-induced redox imbalances that are indicative of oxidative stress in the myometrium. Due to its bulky nature, SOD would not be expected to enter the cultured cells from the treatment medium [38]. This may imply extracellular or plasma cell membrane involvement in the oxidative stress induced by lindane, although, molecules such as SOD may also be internalized from the medium [39, 40].

Membrane lipid peroxidation is a common consequence of cellular oxidative stress. Because lindane exposure elevated thiobarbituric acid (TBA)-reactive substances, the present study suggests that lindane induces lipid peroxidation in myometrial cells. The TBA assay is commonly used to quantify lipid peroxidation and relies upon the reaction of TBA with malondialdehyde, a breakdown product of lipid endoperoxides [41, 42]. Hydroxyl radical attack of membrane phospholipids is a common pathway by which lipid peroxidation occurs [43]. In the present study, cotreatment with D-mannitol, a hydroxyl radical scavenger [20–22], prevented lindane-induced inhibition of Lucifer yellow dye transfer in a concentration-dependent manner. This result suggests that scavenging of hydroxyl radicals may prevent lindane-induced inhibition of gap junctions by interfering with lipid peroxidation. Protective effects of mannitol against inhibition of gap junctional intercellular communication among cultured hepatocytes have been observed by others [35].

Experiments conducted at the tissue level in this study agreed with the findings at the cellular level, showing that lindane eliminated uterine contraction in vitro. As with Lucifer yellow dye transfer studies, lindane's pattern of uterine contractility inhibition was mimicked by the pro-oxidant, tBH. For both lindane and tBH, uterine contractions were decreased over time and were ultimately abolished. The time required for cessation of contractions was slightly longer for tBH compared with lindane. One possible explanation for this difference may be that the more lipophilic nature of lindane allowed it to accumulate more rapidly in the uterine tissue. Neither lindane nor tBH abolished contractions via cytotoxicity because all uterine strips contracted when challenged with KCl following lindane or tBH exposure. Because isolated mid-gestational rat myometrium develops abundant gap junctions in vitro upon incubation [44, 45] and myometrial gap junctional intercellular communication is essential for coordinated uterine contraction [8], it is proposed that lindane-induced oxidative stress disrupted rat uterine contractility as a result of gap junction inhibition.

Because a lindane-induced oxidative stress may increase tissue levels of the reactive species, hydrogen peroxide, there is a possibility that lindane's inhibitory effect is not due to membrane peroxidation, but to altered pH of the cells. Exposure of other cell types to hydrogen peroxide decreases intracellular pH [46]. Furthermore, altered pH can inhibit gap junctional intercellular communication [47–50] and contractility of uterine tissue [51] as well as other muscle [52, 53]. Decreased intracellular pH, in particular, has led to the abolishment of uterine contractions [54, 55]. It should be noted, however, that lindane exposure did not appear to measurably alter extracellular pH of cell cultures or pH of muscle baths throughout the experiments in this study when crudely measured using pH paper strips (data not shown). Nevertheless, this alternative mechanism should be further evaluated particularly because this study did not include accurate measurements of extracellular pH nor measurements of intracellular pH.

Lindane's ability to abolish in vitro uterine contractions was consistent with previous findings from this laboratory [6]. Lindane potently inhibits contractions in other smooth muscle as well, including rat vas deferens [56]. In the present study, pretreatment with {alpha}-tocopherol provided remarkable protection from this lindane-induced inhibition, similar to the protective effects of the antioxidant on lindane's inhibition of gap junctional communication. Although {alpha}-tocopherol was the only antioxidant evaluated in tissue contractility studies, it is speculated that other antioxidants would reverse lindane's inhibition of uterine contractility also, based upon cell responses, and this should be investigated in future work.

{alpha}-Tocopherol protection against lipid peroxidation is well described, and includes scavenging of lipid peroxyl radicals to break membrane-damaging chain reactions [57, 58]. This mechanism of action could explain the protective effects of {alpha}-tocopherol throughout this study. Alternatively, it is possible that the beneficial effects of {alpha}-tocopherol against lindane-induced inhibition of rat uterine function result from prevention of events upstream of the lipid peroxidation caused by lindane. Recently, Wang and Loch-Caruso [59] reported that {alpha}-tocopherol diminished protein kinase C activation of NADPH oxidase, and the subsequent increased generation of superoxide in lindane-exposed rat uterine tissue.

Studies show that lindane bioaccumulates in human adipose tissue [1, 2, 60] as well as uterus and other genitourinary tissue [60–62]. Because fat mobilization during pregnancy would be expected to release general body fat stores into the circulatory system [63, 64] and, subsequently, to tissues such as the uterine smooth muscle, there is potential for increased uterine exposure to lindane during pregnancy due to release from adipose tissue.

Results from this study suggest that exposure to lindane and other pro-oxidants impairs myometrial contraction by inhibiting gap junctional intercellular communication. Should lindane induce oxidative stress and lipid peroxidation in vivo, the possibility of impaired parturition exists. Furthermore, results from this study suggest that this impairment may be prevented by increasing the uterine antioxidant status.

ACKNOWLEDGMENTS

Special thanks to Thea Clipson for technical assistance with the contractility experiments, to Dr. Craig Harris for providing uterine tissue, and to Dr. Kay Criswell for demonstrating use of the microinjection equipment and for first observing lindane's effects on dye transfer in our cultured myometrial cells.

FOOTNOTES

First decision: 3 August 2000.

1 This work was presented in part at the 30th Annual Meeting of the Society for the Study of Reproduction, Portland, Oregon, August 2–5, 1997, and was supported by grant ES06915 from the National Institute of Environmental Health Sciences, NIH, issued to R.L.C.; an institutional predoctoral training grant to T.R.K. (T32-HD07048) from the National Institute of Child Health and Human Development, NIH; and a predoctoral fellowship to T.R.K. from the Horace H. Rackham School of Graduate Studies, University of Michigan. Additional support was provided by the Laboratory Animal Core of the Center for the Study of Reproduction (Grant P30 HD18258 from NICHD, NIH). Contents of the work are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS or NICHD. Back

2 Correspondence: Rita Loch-Caruso, Department of Environmental Health Sciences, M 6108 School of Public Health II, 1420 Washington Heights, University of Michigan, Ann Arbor, MI 48109-2029. FAX: 734 647 9770; rlc{at}umich.edu Back

Accepted: September 19, 2000.

Received: June 26, 2000.

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