Lindane-induced inhibition of spontaneous contractions of pregnant rat uterus

Reproductive Toxicology 13 (1999) 481– 490

Lindane-induced inhibition of spontaneous contractions of pregnant rat uterus Kay A. Criswella, Rita Loch–Caruso* Toxicology Program, Department of Environmental Health Sciences, M6112 School of Public Health II, 1420 Washington Heights, University of Michigan, Ann Arbor, Michigan 48109-2029, USA Received 9 February 1999; accepted 27 June 1999

Abstract Hexachlorocyclohexanes (HCHs) are prevalent insecticides. Lindane (␥-HCH) inhibits uterine gap junctions but ␤-HCH does not. Because gap junctions promote coordination of oscillatory uterine contractions, we hypothesized that lindane, but not ␤-HCH, would inhibit uterine contractions. Uterine strips from midgestation rats were suspended in standard muscle baths and exposed to HCHs in a cumulative manner. Lindane induced concentration-dependent decreases in contraction force (ED50 of 9.2 ␮M) and complete uterine quiescence at 30 ␮M. In contrast, ␤-HCH had no effect on contraction force, but 20 to 200 ␮M ␤-HCH increased contraction frequency in a concentrationdependent manner. Isomer-specific differences in uterine responses were observed at similar HCH isomer tissue concentrations. Additionally, the phospholipase A2 inhibitor and antioxidant quinacrine increased the ED50 for contraction force inhibition to 84.5 ␮M lindane. Lindane also increased cAMP concentrations. Lindane and ␤-HCH have distinctly different actions in the uterus. Lindane’s inhibitory action may involve cAMP, arachidonic acid, or oxidative stress. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Lindane; Inhibition of uterine contraction; Hexachlorocyclohexane; Cyclic AMP; Uterus; Pregnant rat; Pregnant uterus; Quinacrine; Antagonism of lindane action in uterus; Myometrium;

1. Introduction Hexachlorocyclohexane (HCH) is an environmentally persistent pesticide. Its widespread distribution is associated with its long-term usage as an agricultural and forestry insecticide. Although multiple stereoisomers exist, insecticidal activity is largely attributed to the ␥ isomer. This isomer, commonly referred to as lindane, is a current treatment for human and veterinary scabies and lice [1]. Because of its lipophilic nature, adverse effects due to bioaccumulation of HCH have become an increasing concern. Studies have shown the nearly universal presence of various HCH isomers in human fat samples [2]. Lindane is recognized as a neurotoxicant due to the induction of neurologic hyperexcitability in insects and

A portion of this work was presented at the 33rd Annual Meeting of the Society of Toxicology, March 13–17, 1994 (Toxicologist, 14:80, 1994). * Corresponding author. Tel.: (734) 936-1256; fax: (734) 647-9770. a Current address: Parke Davis Pharmaceutical Research, Warner– Lambert Co., 2800 Plymouth Road, Ann Arbor, MI 48105. E-mail address: [email protected] (R. Loch–Caruso)

mammals [3,4]. Lindane also exerts multiple effects on female reproductive function. In the female rat, lindane alters steroid hormone concentrations and multiple reproductive processes [5,6] and exhibits weak estrogenic [7] as well as antiestrogenic actions [8]. Administration of lindane to pregnant rats increased gestation length in one study [9] but no effects on gestation length were evident in a multigeneration study [10]. Lindane potently decreases norepinephrine-induced contractions in rat vas deferens smooth muscle [11]. Forceful, synchronized, oscillatory contractions of uterine smooth muscle are essential for the successful delivery of the fetus. In smooth muscle cells isolated from pregnant rat uteri, lindane stimulates elevations of cyclic adenosine monophosphate (cAMP) [12], a second messenger associated with uterine relaxation [13]. In addition, lindane eliminates gap junctional communication between uterine smooth muscle cells in culture but ␤-HCH has no measurable effect on gap junctions [12]. Because gap junctions likely facilitate coordination of uterine contractions [14], inhibition of gap junctional communication in the myometrium would be expected to impair uterine contractile function. The present

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study examined the hypothesis that lindane, but not ␤-HCH, inhibits spontaneous oscillatory uterine contractions. Additional experiments examined lindane-induced increase of cAMP and quinacrine antagonism of lindane-induced uterine relaxation.

2. Materials and methods 2.1. Chemicals Gamma-hexachlorocyclohexane (lindane, 99% purity), quinacrine, picrotoxin, and bovine serum albumin (BSA) were obtained from Sigma Chemical (St. Louis, MO, USA). ␤-hexachlorocyclohexane (␤-HCH, 98% purity) was obtained from Ultra Scientific (North Kingstown, RI, USA). Stock solutions of 50 mM lindane and ␤-HCH were prepared fresh in dimethyl sulfoxide (DMSO) and diluted in buffer as needed to obtain the final concentration. 2.2. Contractility assessment Spontaneous isometric oscillatory contractions were assessed in uterine muscle strips isolated from pregnant rats using a modified procedure of Juberg et al. [15]. Briefly, midgestation (Day 10) Sprague–Dawley rats were anesthetized with ether and the uteri were removed. The animals were sacrificed by exsanguination while under anesthesia. Embryos and excess fat were removed, and a single longitudinal strip of muscle (2 mm ⫻ 20 mm) was excised from the midsection of each uterine horn. One muscle strip was assigned to the control group and the other strip to the treatment group for each rat. Because it was observed that uterine strips obtained from horns containing less than four implantation sites had irregular or depressed contractile function, rats with less than four implantation sites per horn were excluded from this study. To record isometric contractions, the strips were suspended in 50-mL muscle baths (custom-made by the University of Michigan Glass Shop) containing prewarmed (37°C) physiologic salt solution (PSS; 116 mM NaCl, 21.9 mM NaHCO3, 11.1 mM dextrose, 4.6 mM KCl, 1.16 mM MgSO4 (7H2O), 1.16 mM NaH2PO4(H2O), 1.8 mM CaCl2(2H2O), and 2.6 mM EDTA at pH 7.4). The PSS was supplemented with 25 ␮g/mL BSA to prevent precipitation of the HCHs. The muscle baths and buffer reservoirs were continuously perfused with 95% O2 and 5% CO2. Each experiment consisted of two control strips in one muscle bath and two treated strips in the second muscle bath. One end of each muscle strip was tied with surgical silk to a stationary post whereas the other end was tied to a force transducer (Grass FT-03, Quincy, MA, USA). Contractions were monitored by polygraph. All strips were subjected to a 1.0 g preload tension, allowed to equilibrate at 37°C for 45 min, and then depolarized with a brief exposure to 60 mM KCl to determine maximal contractile force.

Treatment of strips routinely began 2 h after the initial challenge with 60 mM KCl. A pretreatment baseline for frequency and force of contraction was obtained for each strip during the second hour of this equilibration period. A concentration-response experiment was performed by adding lindane at 10-min intervals in a cumulative manner to achieve concentrations of 0.1, 1, 5, 10, 15, 20, and 30 ␮M lindane. Solvent controls were exposed to DMSO alone at concentrations used to deliver lindane (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, and 0.08% DMSO). During the treatment period, contraction frequency was assessed throughout each 10-min exposure period. Because contraction force required 3 to 4 min to stabilize after the addition of lindane, the average contraction force for each strip was calculated during the last 5 min of each exposure period. A contraction was operationally defined as an increase of force that initiated at baseline tension, exceeded 25% of maximal contractile force (as determined by the response to 60 mM KCl), and returned to baseline. At the end of the treatment period, the baths were rinsed 3 or 4 times with prewarmed PSS. Contractions were calculated for the 30 to 60-min period following rinsing. At the end of each experiment, strips were again depolarized with 60 mM KCl to assess muscle viability and response to a depolarizing concentration of KCl. The cumulative concentration response was repeated with ␤-HCH, but in this case using increasing concentrations between 1 and 200 ␮M ␤-HCH. Exposure times and conditions were identical to those described for lindane. In the next set of experiments, longitudinal myometrial strips were allowed to attain spontaneous contractions under conditions previously described. In separate trials, the organ baths were supplemented with the GABAA inhibitor picrotoxin (100 ␮M) or the phospholipase A2 inhibitor and antioxidant quinacrine (10 ␮M). Uterine strips were monitored for contraction frequency and force during a 1-h exposure period. In subsequent experiments, a cumulative concentration-response experiment was performed in the simultaneous presence of each inhibitor and increasing concentrations of lindane. 2.3. Determination of lindane and ␤-HCH concentration in uterine strips Lindane and ␤-HCH concentrations in uterine homogenates were performed by the NIEHS Analytical Core at Michigan State University. Following cumulative additions of lindane, strips were removed from the muscle baths during exposure to 10 ␮M or 30 ␮M lindane and after the bath had been rinsed three times with PSS. Strips treated with similar cumulative additions of ␤-HCH were collected during exposure to 30 ␮M and 200 ␮M ␤-HCH. All strips were weighed, homogenized in PSS, and methanol-extracted using acetonitrile and an octadecyl-bonded silica sorbent prior to being subjected to analysis on a Perkin– Elmer Autosystem gas chromatograph (Norwalk, CT, USA) with a dual column dual electron-capture configuration. A

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wide-bore capillary column (DB-5, J & W Scientific, Folsom, CA, USA) was used to quantify and the second column, a DB-608 (J & W Scientific, Folsom, CA, USA) was used to confirm the identity of the analyte. 2.4. Cyclic AMP assay Specimens were assayed for cAMP activity with the Gibco BRL non-isotopic immunoassay system (Life Technologies, Inc., Grand Island, NY, USA). Essentially, this is a competitive immunosorbent assay for cAMP that utilizes microwell plates precoated with a cAMP-protein conjugate. Uteri from a total of 9 rats were assayed on three separate days. From each of 17 horns, a 2 ⫻ 20 mm strip of longitudinal muscle was excised. Each strip was further cut into four pieces, to be used for each of the four selected treatments, and all pieces were weighed. Each tissue piece was placed in a test tube and exposed to 0.1% DMSO (solvent control), 30 ␮M lindane, 1 ␮M isoproterenol, or 1 ␮M forskolin for 10 min at 37°C. Exposure medium was then aspirated from each test tube and 1 mL of cold 70% ethanol was added. All test tubes were left at 4°C for 10 min to stop the reaction. Each muscle piece was rinsed three times with PSS before adding 1 mL of 0.1 M sodium acetate (pH 6.1). All tissues were homogenized and then centrifuged for 5 min at 1500 g. The supernatant of each tissue was assessed for cyclic AMP activity in duplicate according to the standard method provided by the assay kit manufacturer. The cAMP concentration for each sample was interpolated from a standard curve prepared from a solution of cAMP and normalized per mg muscle tissue. 2.5. Statistical analysis The results are expressed as the mean ⫾ SEM. The ED50 values were calculated as the concentrations of lindane required to inhibit the maximal contraction force by 50%, and were analyzed by one-way analysis of variance (ANOVA) with Prism software (GraphPad Software, San Diego, CA, USA). All other statistical analyses were performed with SigmaStat software (Jandel Scientific, San Fransisco, CA, USA). Analysis of concentration-dependent effects of lindane on contraction frequency and force were analyzed by two-way repeated measures ANOVA with concentration as the repeated measure, applying transformations as needed to normalize the data. Effects of inhibitors were analyzed by twoway repeated measures ANOVA with exposure period as the repeated measure. The cAMP data were analyzed by one-way ANOVA. All post-hoc comparisons of means were by Bonferroni’s multiple comparison test. A P-value ⱕ 0.05 was considered significant.

Fig. 1. Polygraph tracings showing representative uterine activity (a) in a control strip during exposure to the maximal concentration of solvent utilized (0.4% DMSO), (b) during exposure to 15 ␮M lindane (no horizontal bar) and 20 ␮M lindane (horizontal bar), added to the bath in a cumulative manner, (c) following successive rinses (R) with physiologic salt solution after complete relaxation with 20 ␮M lindane exposure, and (d) during exposure to 200 ␮M ␤-HCH. The side bar indicates the unit measures for force (vertical) and time (horizontal) for all tracings.

3. Results 3.1. Concentration-dependent response of uterine contraction to lindane or ␤-HCH The excised uterine segments spontaneously developed oscillatory contractions in the muscle baths. Acute responses to solvent alone (controls), lindane, or ␤-HCH were monitored during 10-min cumulative additions of compound. Representative polygraph tracings obtained from these experiments are shown in Fig. 1. Control strips exposed to solvent alone (maximum concentration 0.4% DMSO) showed regular oscillatory contractions (Fig. 1a). In contrast, Fig. 1b shows that the presence of lindane rapidly and dramatically decreased the amplitude (force) of spontaneous contractions, and that, in this strip, 20 ␮M lindane eliminated oscillatory contractions. Spontaneous oscillatory contractions rapidly resumed within 2 to 3 min after lindane was rinsed from the bath (Fig. 1c), indicating tissue viability as well as rapid reversibility of the effect. In contrast, ␤-HCH failed to inhibit oscillatory contractions at concentrations up to 200 ␮M, but did stimulate increases in the frequency of contraction (Fig. 1d). The relaxant effect of lindane was evidenced by the concentration-dependent depression of contraction force with cumulative additions of the pesticide. Expressed as a percentage of the maximal force observed with 60 mM KCl, no effect on force was observed at 0.1, 0.5, or 1.0 ␮M lindane, but 5, 10, 15, 20, and 30 ␮M lindane inhibited force

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Fig. 2. The effect of lindane, ␤-HCH or solvent alone (controls) on contraction force of uterine strips in muscle baths during cumulative additions of the compounds. Data are expressed as means ⫾ SEM of the percent maximal force achieved with 60 mM KCl (n ⫽ 6 to 14 strips). The asterisks indicate significant inhibition of contraction force in lindaneexposed strips compared to pretreatment (P) (P ⬍ 0.05). Contractile force of lindane-exposed strips returned to normal after rinsing with physiologic salt solution (R). If no error bar is observed, the SEM for that mean is smaller than the size of the symbol.

in a concentration-dependent manner by 27 ⫾ 3.8, 62 ⫾ 7.7, 77 ⫾ 8.0, 95 ⫾ 2.9, and 100%, respectively (Fig. 2; P ⬍ 0.05). In contrast, cumulative additions of ␤-HCH up to 200 ␮M resulted in no loss of contraction force (Fig. 2). After removing the pesticide-containing bath solution and rinsing the tissue three or four times with fresh PSS, oscillatory contractions redeveloped and the force of contractions was indistinguishable among the treatment groups (Fig. 2). The ability of individual uterine strips to generate oscillatory contractions decreased in a concentration-dependent manner, also (Fig. 3). Although all strips continued to oscillate at lower concentrations (ⱕ1 ␮M lindane), oscillatory contractions were observed in only 80% of strips exposed to 5 ␮M lindane. The percent of strips generating oscillatory contractions decreased with increasing concentrations of lindane, and no strips continued oscillating at 30 ␮M. Additionally, no recovery of spontaneous contractions occurred in the continuous presence of 30 ␮M lindane (maximum exposure 1 h). Lindane’s effect on contraction frequency was analyzed also. The frequency of contraction at each concentration (over the 10-min exposure period) was expressed as the percent of baseline pretreatment frequency. The mean frequency of contraction was determined only in strips that continued to exhibit oscillatory contractions during a 10min period (Fig. 4A), to avoid confounding the analysis with effects that were primarily related to force of contraction. Statistical analysis was not performed on these means because lindane decreased the number of contracting strips to very low numbers at the higher concentrations. Fig. 4A shows that lindane’s effect on contraction frequency was

Fig. 3. The percent of uterine strips that continued to exhibit oscillatory contractions during 10-min exposures to increasing concentrations of lindane added in a cumulative manner (n ⫽ 6 strips). Contractions were abolished in all strips at 30 ␮M lindane.

slight and inconsistent, marginally decreasing mean contraction frequency at 5 to 20 ␮M concentrations relative to solvent (DMSO) controls. Although the low number of oscillating strips at the higher concentrations interjects additional uncertainty into any conclusions, it seems that any action lindane may have on contraction frequency is slight compared to its profound effect on the force of contractions. Unexpectedly, ␤-HCH significantly increased contraction frequency in a concentration-dependent manner when compared to solvent (DMSO) controls (Fig. 4B). No effect was observed with 1, 5, 10, or 15 ␮M ␤-HCH. However, contraction frequency increased to 119 ⫾ 4.7, 126 ⫾ 4.3, 129 ⫾ 2.0, 146 ⫾ 12.2, and 166 ⫾ 5.6% with 20, 30, 50, 100, and 200 ␮M ␤-HCH, respectively (P ⬍ 0.05). 3.2. Accumulation of lindane and ␤-HCH in exposed uterine tissue Under conditions identical to the contractility experiments, cumulative additions of lindane at 10-min intervals to 10 ␮M or 30 ␮M lindane resulted in concentrationdependent tissue accumulation of 0.33 ppm and 0.79 ppm lindane, respectively (Table 1). This increase paralleled the chemical’s ability to induce uterine relaxation. Furthermore, rinsing the strips significantly reduced, but did not eliminate, the presence of lindane retained in the strips to 0.15 ppm. Because contractile activity was normal following rinsing, it suggests that 0.15 ppm is below a threshold quantity of lindane required for uterine relaxation. Isolated uterine segments accumulated significantly less ␤-HCH than lindane when exposed to equimolar (30 ␮M) concentrations under equivalent experimental conditions. This result is presumably due to the more limited solubility of ␤-HCH in the aqueous buffer. Nonetheless, exposure to 30 ␮M or 200 ␮M ␤-HCH resulted in tissue residue levels (0.31 and 1.09 ppm, respectively) that were similar to those

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two isomers cannot be explained by differences in solubility or tissue uptake. 3.3. Effect of picrotoxin or quinacrine on lindane-induced uterine quiescence

Fig. 4. Frequency of spontaneous oscillatory contractions in isolated uterine strips exposed to increasing concentrations of (A) lindane or (B) ␤-HCH compared to DMSO (solvent controls). The compounds were added to the muscle bath in a cumulative manner. Data are expressed as means ⫾ SEM (n ⫽ 6 to 13 strips). For the ␤-HCH experiments, the asterisks indicate that contraction frequency during exposure was significantly different from matched DMSO solvent control values (P ⬍ 0.05).

observed with 10 ␮M or 30 ␮M lindane, respectively. Because ␤-HCH stimulated contraction frequency but lindane depressed contraction force at equivalent tissue concentrations, the different uterine responses evoked by the

To examine possible mechanisms of lindane’s action, the acute response to lindane was determined in isolated uterine strips treated with picrotoxin or quinacrine. It has been suggested that lindane acts as an inhibitor of GABAA receptors in neural tissue, resulting in hyperexcitability by preventing Cl⫺ channel closure [4]. Rat uterus has been shown to possess GABAA receptors, and activation of these receptors has been linked to uterine relaxation [16]. To determine if uterine contraction in the in vitro system was responsive to GABAA inhibition, the uterine strips were challenged with picrotoxin, a known GABAA inhibitor. As anticipated, 100 ␮M picrotoxin significantly increased contraction frequency (1.34 ⫾ 0.05 min⫺1) during the treatment period compared to the pretreatment (0.99 ⫾ 0.04 min⫺1) or post-treatment period (0.88 ⫾ 0.05 min⫺1, P ⬍ 0.05; Fig. 5A) without affecting contraction force (Fig. 5B). Because picrotoxin had the opposite effect to that of lindane, it was concluded that lindane inhibition of uterine contraction was most likely not mediated by GABAA receptor inhibition, despite the presence of picrotoxin-sensitive receptors in the uterine strips. The presence of 100 ␮M picrotoxin prior to and concurrent with lindane modestly increased the ED50 for inhibition of contraction force to 14.8 ␮M (Table 2; P ⬍ 0.05), indicating that picrotoxin did antagonize the inhibitory action of lindane slightly. In a prior study in our laboratory, lindane stimulated arachidonic acid release in cultured myometrial smooth muscle cells obtained from pregnant rats [17]. Therefore, the role of phospholipase A2 (PLA2) activation was examined using the PLA2 inhibitor quinacrine [18]. However, quinacrine is also an antioxidant [19,20]. Quinacrine (10 ␮M) alone had no effect on either contraction frequency (Fig. 5A) or the force of contraction (Fig. 5B). However, quinacrine (10 ␮M) strongly antagonized the response to lindane, with a ninefold increase in the ED50 to 84.5 ␮M for strips pretreated with quinacrine (Fig. 6 and Table 2). In

Table 1 Accumulation of lindane and ␤-HCH residues in uterine muscle strips Treatment Lindane Lindane Lindane, followed by 3⫻ rinse with PSS ␤-HCH ␤-HCH

Exposure concentration (␮M) 10 30 30 30 200

Contractile status of uterine strips

Tissue concentration (ppm)a

Significant inhibition of contractile force Quiescent Regular oscillatory activity

0.33 ⫾ 0.06b 0.79 ⫾ 0.09c 0.15 ⫾ 0.03d

Increased frequency of oscillations Increased frequency of oscillations

0.31 ⫾ 0.03b 1.09 ⫾ 0.12c

Values are the means ⫾ SEM for 5 to 7 uterine strips, calculated relative to wet tissue weight. Means with the same superscript letters are not significantly different (P ⬍ 0.05). PSS: physiologic salt solution; ␤-HCH: ␤-hexachlorocyclohexane.

a

b,c,d

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Fig. 6. Quinacrine (10 ␮M) significantly antagonized the inhibitory action of lindane on the force of contraction of isolated oscillating uterine strips (n ⫽ 5). Lindane was added to the muscle baths in a cumulative manner at 10-min intervals. The data are expressed as the mean percent of the maximal KCl-induced contraction ⫾ SEM. Data from Fig. 2, lindane alone, are redrawn here to facilitate comparison with the inhibitor (n ⫽ 14).

Fig. 5. The effect of 100 ␮M picrotoxin or 10 ␮M quinacrine on (A) contraction frequency/min (mean ⫾ SEM) and (B) contraction force (mean ⫾ SEM) expressed as percent of maximal contraction induced by 60 mM KCl. The asterisk indicates that the mean value is significantly different from baseline and post-treatment values for the same treatment group (P ⬍ 0.05). There were 8 strips per treatment group.

addition, 200 ␮M lindane was required to produce quiescence in all strips in the presence of quinacrine.

Because increased cAMP production is associated with uterine relaxation, cAMP levels were measured in lindaneexposed uterine strips. Lindane (30 ␮M) significantly increased cAMP levels in uterine tissue strips (48.4 ⫾ 4 pmol/mg tissue) compared to solvent control strips (5.0 ⫾ 0.7 pmol/mg tissue) (P ⬍ 0.05). Isoproterenol, a compound that increases cAMP through muscarinic receptor activation, and forskolin, a compound that elevates cAMP by direct activation of adenylate cyclase, showed the anticipated increases in cAMP content of 45.2 ⫾ 3 and 152.5 ⫾ 5 pmol/mg tissue, respectively (Fig. 7). 4. Discussion In the process of parturition, contraction of uterine smooth muscle is a highly integrated phenomenon influ-

3.4. Generation of cAMP in lindane-exposed myometrial strips We previously showed that lindane increases cAMP production in cultured myometrial smooth muscle cells [17]. Table 2 Relaxant effect of lindane alone and in combination with picrotoxin or quinacrine Treatment

ED50 (␮M)a

n

Lindane Lindane ⫹ 100 ␮M Picrotoxin Lindane ⫹ 10 ␮M Quinacrine

9.22 ⫾ 1.69 14.80 ⫾ 0.51b 84.48 ⫾ 1.82b

7 5 12

a

Concentration required to inhibit by 50% the maximal contraction force (as induced by 60 mM KCl). Values are mean ⫾ SEM. b Values are significantly different from strips treated with lindane alone (P ⬍ 0.05).

Fig. 7. Concentrations of cAMP generated in uterine strips exposed for 10 min to 0.1% DMSO (solvent control), 1 ␮M isoproterenol, 1 ␮M forskolin, or 30 ␮M lindane. Data are expressed as means ⫾ SEM (n ⫽ 17). Asterisks indicate means that are significantly different from controls (P ⬍ 0.05).

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enced by neuronal, hormonal, metabolic, and mechanical stimulation. Experiments performed in vitro, however, have demonstrated that the myometrium is also myogenic, capable of spontaneous oscillatory contractions. The present study utilized this myogenic characteristic to examine the effects of lindane on rat uterine contractility. Spontaneously oscillating uterine strips exposed to lindane exhibited a rapid, concentration-dependent decrease in force of contraction, with an ED50 of 9.2 ␮M and a concentration of 30 ␮M lindane required for complete uterine quiescence. After removal of the lindane-containing PSS and rinsing with fresh PSS, the uterine strips responded to a depolarizing stimulus of KCl with near-maximal contractile force, indicating that lindane’s relaxation of the uterus was not due to cytotoxicity. The concentration-response relationship was not an artifact of the cumulative nature of the exposure regimen, because uterine strips exposed to single concentrations of lindane exhibited a similar concentrationresponse relationship [21]. ␤-HCH and lindane are stereoisomers of HCH. ␤-HCH was included in these experiments as a structurally similar compound to ascertain whether lindane’s actions in the uterus were of a specific or nonspecific nature. The uterine responses to the HCHs were isomer-specific. Not only did ␤-HCH fail to relax spontaneously contracting uterine muscle, but also the presence of this isomer significantly increased contraction frequency. Similarly, isomer-specific activity of HCHs has been reported in other tissues [22,23]. Because ␤-HCH and lindane evoked divergent uterine responses at similar tissue concentrations, the different responses to the HCH isomers cannot be explained by differences in tissue uptake. Although the focus of this study was on the relaxant effect of lindane, ␤-HCH’s stimulatory effect on uterine contractility may deserve further attention. In humans, bioaccumulation of ␤-HCH typically exceeds that of lindane [24], and high plasma levels of HCH have been associated with preterm birth [25–27]. At concentrations up to 200 ␮M, ␤-HCH has no significant effect on myometrial intracellular calcium concentration [28], ruling out calcium as a potential mediator of ␤-HCH’s uterotonic activity. Although ␤-HCH is weakly estrogenic [29], estrogenic activity is not likely to explain the stimulatory response observed in this experiment because acute exposures to estrogens inhibit contractions in uterus [30,31] and other smooth muscles [32,33]. At the present time, we do not have an explanation for ␤-HCH stimulation of uterine contraction frequency. Additional experiments examined the mechanism by which lindane relaxes the uterus. Lindane inhibits GABAA activated Cl⫺ channels, and it is proposed that lindane induces hyperexcitability in neural tissue through this mechanism [4,22]. However, others have argued against GABAA inhibition as the sole or primary mechanism of lindane’s action [34,35]. The GABA system has been shown to modulate uterine activity, also. In rabbit uterine strips, stimula-

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tion of GABAA receptors inhibits contractile function and stimulation of GABAB receptors enhances contractions [16]. In the present study, the GABAA inhibitor picrotoxin had no effect on contraction force but significantly increased contraction frequency, indirectly indicating the presence of functional GABAA receptors in the uterine tissue. This stimulatory activity was in direct contrast to lindane’s inhibitory effect on uterine contractile force. Although picrotoxin antagonized lindane-induced inhibition of contraction force, the magnitude of the effect was modest. Because the uterine responses to picrotoxin and lindane are in sharp contrast, these results suggest that lindane acts in myometrium by a mechanism different from that of picrotoxin, and that, consequently, lindane does not inhibit uterine contraction by GABAA receptor inhibition. Although ␤-HCH and picrotoxin both stimulated contraction frequency, it is unlikely that the uterotonic activity of ␤-HCH involves GABAA receptor Cl⫺ channel inhibition because ␤-HCH has little or no ability to inhibit the GABAA receptor Cl⫺ channel [22,23]. Activation of PLA2 liberates arachidonic acid from its esterified sn-1,2 position in phospholipids. Metabolism of arachidonic acid results in the production of eicosanoids that include prostaglandins, leukotrienes, hydroperoxyeicosatetraenoic acids (HPETEs), and hydroxyeicosatetraenoic acids (HETEs). In uterine smooth muscle, the predominant prostaglandin produced is prostacyclin [36]. Prostacyclin relaxes vascular smooth muscle [37] and is associated with relaxation of sheep [38] and human [39] uterus. The present study showed that quinacrine, a PLA2 inhibitor, produced a ninefold increase in the lindane concentration (ED50) required to achieve uterine quiescence. This result may suggest that arachidonic acid release is important in lindane’s relaxation of spontaneously contracting uterine strips. However, quinacrine is not a selective PLA2 inhibitor, and may influence uterine contraction by its other actions. In particular, quinacrine can act as a scavenger of superoxide anion [19,40], and preliminary work in our laboratory indicates that other antioxidants effectively reverse lindane’s inhibitory activity in the uterus [41]. Additionally, quinacrine antagonizes prostaglandin E2 stimulation of vascular smooth muscle by an action independent of cyclooxygenase metabolism of arachidonic acid [42]. Although lindaneinduced myometrial relaxation is antagonized by quinacrine, it may be premature to postulate that uterine relaxation is mediated by arachidonic acid release, given the multiple actions of quinacrine. Our previous study showed that lindane increases cAMP production in cultured myometrial smooth muscle cells [17]. Because elevated cAMP is a generally accepted mechanism for uterine relaxation [13], cAMP was measured in lindane-exposed uterine strips. The same concentration of lindane that induced complete uterine quiescence (30 ␮M) also produced a nine-fold increase of cAMP in uterine strips compared to solvent-treated controls. The cAMP response was similar to strips treated with isoproterenol, a ␤-receptor

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agonist and known uterine relaxant [43], but less than onethird of the cAMP level achieved with forskolin, a compound that directly activates adenylate cyclase [44]. These results contrast with reports that lindane inhibits agonistinduced accumulations of cAMP in rat enterocytes [45] and rat renal tubules [46], suggesting that lindane’s actions on cAMP may be tissue-specific or may be modified by agonist activation. Although we did not measure lindane-induced cAMP production in the presence of quinacrine, others have reported that quinacrine reduces cAMP production in some tissues [47,48] but has no such effect in other tissues [49, 50]. Thus, although it is possible that quinacrine antagonized lindane’s inhibition of uterine contraction force by preventing increased cAMP production, further investigation is required to determine if these actions are mechanistically linked. Lindane exerted no significant effect on contraction frequency, suggesting that pacemaker activity was unaffected. In contrast, the force of contraction was decreased in a concentration-dependent manner. Hirst and Neild [51] demonstrated that the magnitude of smooth muscle contraction is associated with the number of cells synchronously activated. Gap junctions are hypothesized to play an essential role in the synchronization of uterine contractions by enabling the electrical and metabolic coupling of myometrial smooth muscle cells. Rapid closure of gap junctions would limit the number of synchronized cells, thereby decreasing the force of contraction of the uterine tissue. Although the exact mechanism by which lindane induces uterine relaxation cannot be determined by these experiments, it is interesting to note that lindane rapidly eliminates gap junctional communication in cultured rat myometrial myocytes at concentrations as low as 10 ␮M [12], similar to the effective concentrations observed in the present study. Consistent with the lack of inhibition of uterine contraction by ␤-HCH in the present study, no inhibition of myometrial gap junctional communication was observed with ␤-HCH in a previous study [12]. If lindane eliminates gap junctional communication in the myometrium as it does is in cultured myometrial cells, this would likely limit propagation of action potentials and may thereby contribute to the inability to maintain forceful and oscillatory contractions. The concentrations used in this study, 0.1 to 30 ␮M lindane and 200 ␮M ␤-HCH, exceeded by one to two orders of magnitude the serum concentrations observed in general populations of pregnant women [52,53]. Unfortunately, the lindane concentrations measured in uterine strips in the present study (0.33– 0.79 ppm relative to tissue wet weight) cannot be directly compared to the uterine concentration reported in pregnant women (2 ppm in extracted lipids) [53] because our tissue samples were too small to measure extracted lipid weight. Uterine dysfunction is a relatively common complication of parturition. Post-term delivery (after 42 weeks gestation) occurred in 8.7% of live births in the US in 1996 [54]. Dysfunctional labor (failure to progress in a normal pattern

of labor) occurred in 2.75% of live births and prolonged labor (abnormally slow progress of labor exceeding 20 h) occurred in 0.9% of live births in the US in 1996 [54]). It remains unclear whether bioaccumulation of environmental toxicants such as lindane may be contributing to these complications of parturition. Notably, the tissue distribution of several organochlorine insecticides, including HCHs, is altered during pregnancy. In particular, HCH concentrations in lipids extracted from the uteri of pregnant women were threefold and fivefold higher than in lipids extracted from adipose tissue or maternal blood, respectively [53]. This is markedly different from the nonpregnant state, and suggests that the uterus may be exposed to higher concentrations of such chemicals during pregnancy. The present study clearly shows that lindane inhibits the force of uterine contraction. Moreover, this effect is isomerspecific, because ␤-HCH stimulates contraction frequency. Although the mechanism of lindane’s action remains unresolved, the antagonism of lindane’s activity by quinacrine and stimulation of cAMP generation by lindane suggest that second messenger systems may be mechanistically involved in lindane-induced uterine relaxation. Current experiments in our laboratory are exploring these possibilities. Acknowledgments This research was supported by grants to R.L.–C. (R01ES06915, R29-ES04424, and P42-ES04911), and by an institutional predoctoral training grant to K.A.C. (ES07062) from the National Institute of Environmental Health Sciences (NIEHS), NIH. Additional support was provided by the Laboratory Animal Core of the Center for the Study of Reproduction (NIH P30-18258). Vincent Peterkin assisted with graphic presentations and statistical analysis, Dr. Craig Harris donated rat uteri, Dr. Daland Juberg first observed lindane’s action on uterus, Dr. Paul Loconto of the Michigan State University NIEHS Analytical Core measured lindane and ␤-HCH in tissue, Dr. Mei–Ling Tsai provided technical assistance, and Mrs. Ruby Patterson provided secretarial assistance. References [1] Smith AG. Chlorinated hydrocarbon insecticides. In: Hayes WJJ, Laws ERJ, eds. Chlorinated hydrocarbon insecticides, vol. 2. San Diego: Academic Press; 1991:731–915. [2] Robinson PE, Mack GA, Remmers J, Levy R, Mohadjer L. Trends of PCB, hexachlorobenzene, and beta-benzene hexachloride levels in the adipose tissue of the US population. Environ Res 1990;53:175– 92. [3] Narahashi T. Effects of insecticides on nervous conduction and synaptic transmission. In: Wilsinsons CF, ed. Effects of insecticides on nervous conduction and synaptic transmission. New York: Plenum Press; 1976:329 –52. [4] Ogata N, Vogel SM, Narahashi T. Lindane but not deltamethrin blocks a component of GABA-activated chloride channels. FASEB J 1988;2:2895–900.

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