Toxicity of Cadmium in Human Trophoblast Cells (JAr Choriocarcinoma): Role of Calmodulin and the Calmodulin Inhibitor, Zaldaride Maleate

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

144, 225– 234 (1997)

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Toxicity of Cadmium in Human Trophoblast Cells (JAr Choriocarcinoma): Role of Calmodulin and the Calmodulin Inhibitor, Zaldaride Maleate Stephanie Schubach Powlin,* Peter C. Keng,† and Richard K. Miller*,1 *Department of Obstetrics and Gynecology, Department of Environmental Medicine, and †Department of Radiation Oncology, University of Rochester, Rochester, New York 14642 Received July 18, 1996; accepted February 10, 1997

Toxicity of Cadmium in Human Trophoblast Cells (JAr Choriocarcinoma): Role of Calmodulin and the Calmodulin Inhibitor, Zaldaride Maleate. Powlin, S. S., Keng, P. C., and Miller, R. K. (1997). Toxicol. Appl. Pharmacol. 144, 225 – 234. Cadmium (Cd), the heavy metal, is toxic to the placenta. The objectives of this study were to determine if Cd toxicity is due to inhibition of placental or trophoblast cell proliferation through interactions with the intracellular calcium binding protein, calmodulin (CaM). Cd can replace calcium and thus interfere with CaM’s function. Also, CaM inhibitors reverse selected toxic effects of Cd. The CaM inhibitor, zaldaride maleate, was used to determine if Cd inhibits trophoblast cell proliferation through interactions with CaM. JAr choriocarcinoma cells, a neoplastic trophoblast cell line which is similar to early human trophoblast cells, were selected to study this question. Cd (20 and 40 mM) inhibits JAr cell proliferation, as measured by cell number and BrdU incorporation. Zaldaride (10 and 20 mM) inhibits proliferation to a lesser extent; 100 mM is lethal. To determine if zaldaride alters actions of Cd, zaldaride and Cd are added simultaneously. Zaldaride (20 mM) and Cd (20 mM) together inhibit proliferation less than Cd alone, thus partially protecting cells. Metallothionein is induced in cells exposed to Cd, while zaldaride does not cause induction of this cellular defense mechanism protein. To determine if Cd inhibits proliferation through alterations of cell cycle, JAr cells enriched for G0/G1 phase were exposed to 20 mM Cd, 20 mM zaldaride, or 20 mM Cd plus 20 mM zaldaride for 24 hr. Cells remain in G0/G1 following Cd exposure; cells treated with 20 mM zaldaride progress through S phase and into G2. Zaldaride and Cd together allow JAr cells to leave G1 and enter S phase, partially relieving the cycle block produced by Cd. This study demonstrates a role for calmodulin in mediating the toxicity of Cd in trophoblast cell proliferation. q 1997 Academic Press

The heavy metal cadmium (Cd) is toxic to placentae from both animals and humans. Parizek (1964) first demonstrated the placental necrosis and fetal death which accompanies 1 To whom correspondence should be addressed at University of Rochester Medical Center, Department of Obstetrics and Gynecology, 601 Elmwood Avenue —Box 668, Rochester, NY 14642-8668.

maternal exposure to Cd at term in the rat. Cd accumulates in the placentae of rats, hamsters, and mice, leading to teratogenesis and fetal death (Chiquoine, 1965; Wolkowski, 1974; Dencker, 1975; Gale and Layton, 1980). Levin and associates (Levin and Miller, 1980; Levin et al., 1981) demonstrated that the rat fetotoxicity observed following exposure to Cd was a direct nonvascular effect of Cd on the placenta. Using a human placental perfusion model, Wier and coworkers (1990) demonstrated that exposure to Cd (10, 20, or 100 nmol/ml) produced ultrastructural changes (subsyncytiotrophoblastic vesiculations, stromal edema, and vacuoles in placental macrophages) in term placental tissue, with necrosis occurring at 100 nmol/ml within 6 –8 hr. Biochemical parameters for placental function were also inhibited by Cd, as production of human chorionic gonadotropin (hCG) was decreased by 20 and 100 nmol/ml Cd. hCG is produced by the syncytiotrophoblast. The human placenta is composed of two distinct placental (trophoblast) cell types. The villous cytotrophoblasts (CT) either fuse and form the outer layer of multinucleated syncytiotrophoblasts (ST) or proliferate at the villous tip (CT cell column) and become invasive extravillous trophoblasts (EVT). Besides hCG, ST cells are responsible for the synthesis of many other hormones such as human placental lactogen (hPL), PAPP-A, estriol, and progesterone (Miller and Thiede, 1984). Women may be exposed to Cd in the workplace (welding and battery manufacture) as well as via cigarette smoke. Placental levels of Cd are increased in women who smoke during pregnancy (Roels et al., 1978; Kuhnert et al., 1982). There is a concern that pregnant women exposed to Cd may experience an increase in adverse pregnancy outcomes. Case reports have associated women exposed to Cd in the workplace with an increase in early pregnancy loss (Eisenmann and Miller, 1996). Additionally, animal studies demonstrate that early exposure to Cd may cause impaired reproduction (Kreis et al., 1993). We hypothesize that the placental toxicity and/or early pregnancy loss induced by Cd may be due to inhibition of trophoblast cell proliferation. A crucial step in establishing

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pregnancy is implantation of the placenta into the uterine endometrium. This invasive process involves the proliferation and differentiation of CT. Cd inhibits cell proliferation in other cell types (Kaji et al., 1993; Piersma et al., 1993; Cifone et al., 1989), yet the mechanism is not clearly understood. Does Cd inhibit trophoblast cell proliferation? Cd could inhibit cell proliferation via many different mechanisms, some of which may include general cytotoxicity, altering regulators of the cell cycle (cyclins and cyclin dependent kinases), inhibiting energy metabolism, or modifying other metals and metal binding proteins (e.g., zinc, metallothionein, calcium, and calmodulin). Cd is most likely inhibiting cell proliferation through interference with metal binding proteins such as metallothionein and calmodulin. Induction of metallothionein is associated with protection against Cd toxicity (Klaassen and Suzuki, 1991). Although Cd can induce metallothionein in the human placenta, acute Cd toxicity is still exhibited (Breen et al., 1994; Powlin et al., 1994). This suggests that there may not be sufficient or rapid enough induction to protect against the concurrent exposure to Cd. We, therefore, hypothesized that Cd may decrease trophoblast cell proliferation through interactions with the intracellular calcium binding protein, calmodulin (CaM). CaM is important in many processes involved in cell proliferation (Hidaka et al., 1981; Chafouleas et al., 1982; Cheung, 1984; Takuwa et al., 1993; Colomer et al., 1994). Cd can replace calcium, inducing a similar conformational change in calmodulin and thus remove the control of calmodulin from calcium (Cheung, 1988), which could disturb many cellular functions. CaM inhibitors ameliorate selected toxic effects of Cd such as testicular toxicity (Niewenhuis and Prozialeck, 1987) and microtubule disassembly (Perrino and Chou, 1986). We used the calmodulin inhibitor, zaldaride maleate, alone and in combination with Cd, to examine the interaction of Cd with CaM and the possible role in regulating trophoblast cell proliferation. Zaldaride has an advantage over other calmodulin inhibitors because it is potent and, more importantly, does not inhibit protein kinase C activity at doses up to 100 mM (Norman et al., 1987). JAr choriocarcinoma cells, a trophoblast cell line having many characteristics similar to first trimester trophoblast cells, were used to explore these questions. JAr cells are rapidly proliferating, invasive, and synthesize placental hormones such as hCG and progesterone (Pattillo and Gey, 1968). JAr cells enabled us to examine the effects of Cd and zaldaride maleate, alone and in combination, on cell proliferation as well as cell cycle progression. MATERIALS AND METHODS JAr cell counting experiments. JAr cells (provided by Dr. R. A. Pattillo, Atlanta, GA) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD), 1% glutamine (Gibco), and 1% penicillin/streptomycin (Gibco). Flasks were seeded with 2 1 105

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JAr cells. Day 1 and Day 2 after seeding, two control cultures (attached and floating cells) were counted using a hemacytometer. Cd, as CdCl2 , was diluted in medium to 0.5, 2, 20, or 40 mM. Zaldaride maleate (1,3-dihydro1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate) (generously donated by Dr. Peter Fels, Zyma, Nyon, Switzerland) was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM and then diluted in RPMI 1640 to final concentrations of 10, 20, or 100 mM. In experiments where Cd and zaldaride were added together, medium containing Cd was made first and then zaldaride was added. On Day 2, medium containing Cd, zaldaride, or both Cd and zaldaride was added and removed after 24 hr (Day 3). On Day 3 and every day thereafter, through Day 6, one culture each from control and treated groups was counted (attached and floating cells). Floating cells were characterized by the lack of attachment to the culture flask (they were removed with media) and irregularly shaped cell membranes. Cell viability was determined using Trypan blue dye exclusion. Briefly, two drops of 0.2% trypan blue (in 11 Hanks’ balanced salt solution) were added per milliliter of cell suspension or culture medium with floating cells. Four counts were made from each flask and the numbers were averaged. Medium was saved daily and frozen for biochemical analyses. hCG and progesterone were measured using the ImmuLite assay (Diagnostic Products Corp., Los Angeles, CA) to clinical quality assurance standards. The number of floating cells was expressed as a percentage of the total number of cells. hCG and progesterone were calculated as amount of hormone synthesized per cell before conversion to percent of control values. Centrifugal elutriation/flow cytometry. JAr cells (1 1 106) were seeded into 225-cm2 culture flasks (12 total) and allowed to grow for 6 days. At the end of the culture period, cells were stripped from the flasks and triturated to attain single cell suspensions. Cells were then spun at 1000 rpm for 3 min and resuspended in 20 ml RPMI 1640 medium supplemented with 1% penicillin/streptomycin, 1% glutamine, and 20% fetal calf serum (Gibco). Cells were filtered through a sterile 100-mm wire mesh grid to remove any debris, and then approximately 10 mg of DNase (Sigma) was added to prevent cells from clumping together. Cells were rocked on an orbital rocker for 1 hr prior to elutriation. JAr cells (5 1 107 to 2 1 10 8 in 20 ml) were loaded into a Beckman (Model J-6M) Induction Drive Centrifuge at 4 7 C, a flow rate of 28 ml/ min, and a speed of 3250 rpm. Elutriation medium consisting of RPMI 1640 supplemented with 1% penicillin/streptomycin and 5% newborn calf serum (Atlanta Biologicals, Norcross, GA) was used to collect all cell fractions. Two 40-ml samples (A and B) were withdrawn at each rotor speed. Rotor speeds were decreased from 3250 to 2100 rpm. Cell counts, diameter, and volume were calculated on each ‘‘B’’ sample using a Coulter Channelyzer 256. Cells from the G0/G1 phase of the cell cycle were collected. The separation was based on the Coulter data of volume; G0/G1 cells were approximately 1500 m3. Cells from freshly elutriated samples (at least 5 1 105 ) were fixed in 70% ethanol overnight, and then cellular DNA was stained with propidium iodide to determine the stage in the cell cycle. Cells were analyzed using the Coulter Epics Profile flow cytometer (Coulter Corporation, Hialeah, FL). Cells (1– 2 1 106 ) from the enriched G0/G1 population were seeded into large culture flasks, allowed to attach for 2 hr, and then 20 mM Cd, 20 mM zaldaride, or 20 mM Cd plus 20 mM zaldaride was added for 24 hr. Cells were then fixed, stained with propidium iodide as described above, and analyzed by flow cytometry. Determinations of distribution within the cell cycle were made using a multi-Gaussian cell cycle analysis model. To assess DNA synthesis in control and Cd-treated JAr cells, incorporation of the thymidine analogue, bromodeoxyuridine (BrdU) was measured using flow cytometry. JAr cells (3 1 106) were seeded and allowed to grow for 2 days. Cd (20 mM) was added to half the flasks for 24 hr; the corresponding controls were grown in normal medium. During the last 2 hr of culture, 10 mM BrdU was added to all flasks. Cells were then fixed for flow cytometry

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CADMIUM TOXICITY IN JAr CELLS—ROLE OF CALMODULIN analysis. Dual staining using anti-BrdU antibody (Boehringer-Mannheim, Indianapolis, IN) and propidium iodide was performed. Determinations of the percent of BrdU labelled and unlabelled S-phase cells were made using the Cytologics program (Coulter Corp.). Immunocytochemistry. JAr cells (5 1 104) were seeded into fourchamber glass Lab Tek slides (Nunc, Naperville, IL) and allowed to grow for 2 days. Cd (20 mM), zaldaride (20 mM), or a combination was added for 24 hr. Medium was then aspirated, and cells were washed briefly with phosphate-buffered saline (PBS), and then fixed for 15 min in 10% formalin. After washing again with PBS, cells were solubilized for 15 min in 1% Nonidet P-40 (in PBS). Cells were then washed, and immunocytochemistry was performed according to the Vectastain Elite ABC kit protocol (Vector Laboratories, Burlingame, CA). Metallothionein (MT) antibody (clone E9, Dako) was used in a 1:40 dilution for 1 hr. Statistics. Data were analyzed using a three-way, mixed model analysis of variance. The three factors were treatment (dose), time (day), and experiment. The last of these was included because the study was performed in replicate experiments; this factor was treated as a random effect. Each analysis included an examination of residuals as a check on the required assumptions of normally distributed errors with constant variance. The analysis focused on the effects of the various treatments. If the treatment by time interaction was significant, it was concluded that differences between treatments were changing over time. In this case separate two-way analyses of variance were performed for each day. These included all pairwise comparisons of the treatment means using the Tukey method of multiple comparisons, with a 5% overall error rate. If the treatment by time interaction was not significant in the full analysis, then the comparison of the treatments was based on the treatment effect (main effect) in this analysis.

RESULTS

Cell Proliferation and Hormone Synthesis/Release We hypothesized that Cd-induced placental toxicity may be due, in part, to an inhibition of trophoblast cell proliferation. Cd (20 and 40 mM) decreased the number of attached JAr cells in a dose-related manner (Fig. 1A). The lower doses of Cd (0.5 and 2 mM) did not significantly differ from controls. Cd (20 and 40 mM) also increased the percentage of floating cells in a dose-dependent manner (Fig. 1B). Floating cells were considered to be dead if trypan blue dye was intracellular. Control JAr cells have a doubling time of approximately 24 hr, which is supported by the current data (21.5 { 4.6 hr). Lower concentrations of Cd (0.5 and 2 mM) did not alter this rate, while 20 mM increased the time to 45.2 { 16 hr, and 40 mM appeared to completely halt proliferation (Fig. 1A). Exposure to Cd (20 mM) decreased BrdU incorporation by JAr cells. All S-phase cells in control cultures incorporated BrdU (100%), compared to only 69.3 { 0.1% of Cd-treated cells. Higher concentrations of Cd (20 and 40 m M) increased the amount of hCG synthesis per cell on Day 5 and Day 6 (Fig. 2A). Progesterone demonstrated a similar trend, with higher concentrations of Cd increasing synthesis (Figure 2B). One mechanism by which Cd could inhibit cell proliferation is through interactions with calmodulin. We, therefore,

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used a calmodulin inhibitor, zaldaride maleate, to examine these possible interactions and the effect of JAr cell proliferation. Zaldaride decreased the number of attached JAr cells (Fig. 3), with 10 and 20 mM zaldaride inhibiting proliferation similarly to 2 mM Cd. Zaldaride (10 and 20 mM) did not affect the doubling time of JAr cells (Figs. 3 and 4A). However, 100 mM zaldaride, like 40 mM Cd, halted proliferation (Fig. 4A). Only 100 mM zaldaride increased the number of floating cells compared to control cultures (Fig. 4B). To determine if zaldaride protected cells from the inhibition of proliferation caused by Cd, zaldaride and Cd were added simultaneously. The addition of 20 mM zaldaride with 20 mM Cd partially protected JAr cells, as seen by an increase in the number of attached cells, when compared to treatment with Cd alone (Fig. 5A); these numbers were significantly different after Day 4. Zaldaride and Cd together decreased the doubling time compared with Cd alone (27 { 13.1 hr versus 45.2 { 16.0 hr; p õ 0.05) (Fig. 5A). The addition of Cd and zaldaride decreased the percentage of floating cells compared to Cd alone on Days 4 and 5 (Fig. 5B). Cadmium (20 mM) exposure for 24 hr on Day 2 has a differential effect on hCG and progesterone synthesis. The hCG production is inhibited by Cd on Day 3 and has a recovery such that by Day 5 the Cd-treated cells have significantly increased hCG synthesis/release (Fig. 6A). In contrast, progesterone synthesis/release is significantly higher in Cd-treated cells than in controls on every day of culture (Fig. 6B). When Cd and zaldaride are added simultaneously, zaldaride does not reverse the effects of Cd on hCG and progesterone synthesis/release. Compared to controls, hCG synthesis is inhibited by the combination on Day 3 and does not differ from Cd alone throughout the culture period (Fig. 6A). It was determined, following three-way ANOVA, that progesterone synthesis/release by the three treatments (Cd, zaldaride, and Cd plus zaldaride) did not significantly differ from each other, but Cd-induced progesterone synthesis was significantly higher than control levels (Fig. 6B). Zaldaride alone does not alter hCG synthesis/release from control levels on 3 of 4 days (Fig. 6A), and progesterone synthesis/release does not differ from controls throughout the culture period (Fig. 6B). In conclusion, Cd produces significant and different responses for both hCG and progesterone production, zaldaride exposure does not differ from controls, and the combination of cadmium and zaldaride does not differ from Cd alone. Thus, zaldaride does not adversely affect protein and steroid hormone production by JAr cells, nor does it reverse the effect of Cd. Flow Cytometry Unsynchronized JAr cells exposed to control conditions or 20 mM Cd for 24 hr did not differ in their cell cycle

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FIG. 1. (A) Effect of cadmium on JAr cell proliferation rate. (B) Effect of cadmium on ‘‘floating’’ JAr cells. Cells were exposed to Cd on Day 2 for 24 hr. Values represent means of five experiments. Standard deviations deleted for clarity. Actual number of floating cells in controls: Day 3, 12285 { 3330; Day 4, 18000 { 9530; Day 5, 49959 { 52930; Day 6, 44375 { 20570. m, significantly different from control, 0.5 Cd and 2 mM Cd; /, significantly different from all lower Cd doses and control.

distribution (data not shown). Using centrifugal elutriation, we attained 71% G0/G1 cells in the enriched population (Fig. 7, Table 1). When these cells were allowed to progress through the cell cycle as a control, after 24 hr there were

40% in G0/G1, 44% in S, and 16% in G2/M (Fig. 7, Table 1). Treatment with 20 mM Cd for 24 hr caused cells to remain in G0/G1 (82% in G0/G1 and 18% in S) compared to controls. Zaldaride (20 mM) allowed cells to progress

FIG. 2. (A) Effect of cadmium on hCG synthesis by JAr cells. (B) Effect of cadmium on progesterone synthesis by JAr cells. Cells were exposed to Cd on Day 2 for 24 hr. Values in both figures represent means { SD of five experiments. Data are expressed as percentages of corresponding control values. hCG control values expressed as mIU per cell: Day 3, 0.029 { 0.016; Day 4, 0.027 { 0.012; Day 5, 0.020 { 0.008; Day 6, 0.013 { 0.005. Progesterone control values expressed as pg per cell: Day 3, 0.126 / 0.046; Day 4, 0.156 { 0.065; Day 5, 0.114 / 0.058; Day 6, 0.069 / 0.034. ∗, significantly different from control; s, significantly different from 2 mM Cd; significantly different from 0.5 mM Cd; /, significantly different from all lower Cd doses and control.

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FIG. 3. Effect of zaldaride maleate on JAr cell proliferation. Cells were exposed to Cd on Day 2 for 24 hr. Data are expressed as percentages of corresponding control cell numbers. Values represent means { SD of the following number of experiments (in parentheses): 10 mM zaldaride (4), 20 mM zaldaride (3), 100 mM zaldaride (2). Actual number of control cells: Day 3, 576180 { 690520; Day 4, 1121080 { 1294740; Day 5, 1930140 { 2009620; Day 6, 3177190 { 2638930. ∗, significantly different from control; ❖, significantly different from all groups.

through the cell cycle, but at a different rate than control cells (26% in G0/G1, 57% in S, and 17% in G2/M). When cells were treated with 20 mM Cd and 20 mM zaldaride, more cells were able to leave G0/G1 and enter S than when cells were treated with Cd alone (52% in G0/G1, 47% in S, and 1% in G2/M).

Metallothionein Immunocytochemistry JAr cells were stained immunocytochemically with antiMT antibody to determine protein expression and localization in cells treated with Cd, zaldaride, or a combination of the two. Control JAr cells demonstrated MT protein associ-

FIG. 4. (A) Effect of zaldaride maleate on JAr cell proliferation rate. Cells were exposed to Cd on Day 2 for 24 hr. (B) Effect of zaldaride maleate on ‘‘floating’’ JAr cells. Values in both figures represent means of the following number of experiments (in parentheses): control (4), 10 mM zaldaride (4), 20 mM zaldaride (3), 100 mM zaldaride (2). Standard deviations deleted for clarity. Actual number of floating cells: Day 3, 19375 { 13090; Day 4, 17080 { 5640; Day 5, 21875 { 11000; Day 6, 30625 { 13010. ∗, significantly different from control; ❖, significantly different from all groups.

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FIG. 5. (A) Effect of cadmium and zaldaride maleate on JAr cell proliferation rate. (B) Effect of cadmium and zaldaride maleate on ‘‘floating’’ JAr cells. Cells were exposed to Cd on Day 2 for 24 hr. Values in both figures represent means (n Å 3). Standard deviations deleted for clarity. Actual number of floating cells: Day 3, 23540 { 9970; Day 4, 33330 { 11610; Day 5, 68750 { 29810; Day 6, 119790 { 51480. ∗, different from control; l, significantly different from 20 mM Cd; ❖, significantly different from all groups.

ated almost solely within the nucleus (Fig. 8A). Staining of nuclei is heterogenous, with a few being negative. Treatment of cells with 20 mM Cd for 24 hr resulted in an increase in

protein levels (noted by intensity of staining), as well as a change in the localization (Fig. 8B). MT protein is still associated with the nucleus, but a dramatic increase in cytosolic

FIG. 6. (A) Effect of cadmium and zaldaride on hCG synthesis by JAr cells. (B) Effect of cadmium and zaldaride on progesterone synthesis by JAr cells. Cells were exposed to Cd on Day 2 for 24 hr. Values in both figures represent means { SD (n Å 3). Data expressed as percent of corresponding control values. hCG control values are expressed as mIU hCG per cell: Day 3, 0.017 { 0.001; Day 4, 0.015 { 0.003; Day 5, 0.016 { 0.008; Day 6, 0.015 { 0.011. Progesterone control values expressed as pg per cell: day 3, 0.099 { 0.016; Day 4, 0.071 { 0.016; Day 5, 0.061 { 0.014; Day 6, 0.050 { 0.006). ∗, significantly different from control; h, significantly different from 20 mM zaldaride; l, significantly different from 20 mM Cd.

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FIG. 7. Effect of cadmium and zaldaride maleate on JAr cell cycle progression. X-axis, relative amount of DNA; Y-axis, relative amount of cells; Z-axis, experimental group. Cells were exposed to Cd, zaldaride, or both for 24 hr on Day 2 of culture. Groups are arranged to better visualize cell distributions. Data represent means of the following number of experiments (in parentheses): control (12), G1 (15), 20 mM Cd (12), 20 mM zaldaride (6), 20 mM Cd / 20 mM zaldaride (3).

MT is seen. Cells that are treated with 20 mM zaldaride are identical to control cells, with MT being localized to the nucleus (Fig. 8C). When the combination of Cd and zaldaride is added, cells express MT protein similar to Cd alone (Fig. 8D). Cells exposed to both Cd and zaldaride have MT associated with both the nucleus and cytoplasm. Thus,

TABLE 1 Effect of Cadmium and Zaldaride (Alone and Together) on JAr Cell Cycle Progression Group

N

Unsynchronized G1 Control 20 mM Cd 20 mM zaldaride Cd / zaldaride

15 15 12 12 6 3

%G0/G1 38.6 71 40.2 82.4 25.7 51.9

{ { { { { {

3.3 6.7 11.5 6.6b 6.5a,b,c 2.9c,d

%S 49.8 28.7 43.5 17.5 56.7 47.4

{ { { { { {

3.2 6.8 5.1a 6.6b 5.3a,b,c 2c

%G2/M 11.6 0.3 16.3 0.1 17.6 0.7

{ { { { { {

3.8 0.6 7.9 0.2b 9.2c 1.3b,d

Note. Means { SD. a Different from unsynchronized cells (p õ 0.05). b Different from control ( p õ 0.05). c Different from Cd (p õ 0.05). d Different from zaldaride (p õ 0.05).

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zaldaride alone does not induce MT, nor does it inhibit the induction of MT by Cd. DISCUSSION

Cadmium inhibits, in a dose-related manner, JAr cell proliferation, as measured by cell number, BrdU incorporation, doubling time, and JAr cell cycle progression. JAr cells normally have a doubling time of approximately 24 hr. The two higher doses of Cd (20 and 40 mM) slowed the proliferation rate of JAr cells or, alternatively, induced cells to leave the cell cycle and enter G0. The latter hypothesis is consistent with our observation in elutriated JAr cells that Cd-treated cells accumulate in G0/G1. The incorporation of BrdU into only some of Cd-treated S-phase cells also supports the hypothesis that a small percentage of Cd-treated cells leave the cell cycle. Cd (20 and 40 mM) also significantly increased the number of floating (dead) cells. It appears that Cd initially exerts a cell killing action, with cells surviving the insult demonstrating a decrease in the doubling time. Cd does not inhibit hormone synthesis; in fact, hCG and progesterone syntheses are increased in cells treated with 20 and 40 mM Cd. In response to Cd, JAr cells treated with these doses of Cd appear to have shifted from proliferation

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FIG. 8. Metallothionein immunostaining (clone E9) of JAr cells. (A) Control cells, (B) Cd-treated cells (20 mM), (C) zaldaride-treated cells (20 mM, (D) Cd (20 mM) and zaldaride (20 mM)-treated cells. All cells were exposed for 24 hr (Day 2 to Day 3) and then fixed and evaluated. Positive staining is black. Bar, 50 mm. Note the nuclear localization in control and zaldaride treated cells. Cells stain heterogenously, with a few scattered cells being MT negative (∗). Cd-treated cells demonstrate extensive staining not only in the nucleus, but also in the cytoplasm. Cells treated with both Cd and zaldaride exhibit similar staining to Cd-treated cells.

to increased hormone synthesis. Other agents, such as actinomycin D, methotrexate, and hydroxyurea, which are toxic to choriocarcinoma cells (inhibit DNA synthesis), also increase hCG synthesis (Hussa et al., 1973; Arbiser et al., 1991). It has been suggested that DNA synthesis inhibitors arrest choriocarcinoma cells at a phase of the cell cycle where hormone production is highest (Azizkhan et al., 1979). Cd may inhibit cell proliferation via different mechanisms. Cd does not appear to alter proliferation through general cytotoxicity, nor through changes in energy metabolism (Malek et al., 1996). Cd also probably does not inhibit proliferation through direct interactions with cyclins or cyclindependent kinases, as there is no evidence for metal binding sites on any of these proteins. It was hypothesized that Cd inhibits cell proliferation through interactions with metal-

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binding proteins. To explore the possibility that Cd may inhibit cell proliferation through interactions with CaM, the highly specific CaM inhibitor, zaldaride maleate, was used both alone and in combination with Cd. Zaldaride has been investigated in both animal studies and human clinical trials as an antidiarrheal agent (Shook et al., 1989; DuPont et al., 1993). Unfortunately, these studies don’t provide the blood concentrations of the drug which were effective at treating this ailment. We were, therefore, unable to comment on the therapeutic relevance of the doses of zaldaride used in these experiments. Zaldaride exposure alone (10, 20, and 100 mM) for 24 hr inhibited JAr cell proliferation as measured by cell numbers. When Cd and zaldaride were added together, a partial protection from the inhibition of cell proliferation produced by Cd alone was

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CADMIUM TOXICITY IN JAr CELLS—ROLE OF CALMODULIN

observed. Cd-treated cells have an increase in the doubling time, while zaldaride-treated cells do not differ from control. The combination of Cd and zaldaride significantly increases the number of cells compared to the Cd-treated group on Days 5 and 6, such that the numbers did not differ from zaldaride alone. Zaldaride significantly decreased the doubling time of JAr cells exposed to Cd from 48 to 30 hr. Cadmium alone induced elutriated JAr cells to remain in G0/G1 when compared to control cells. Zaldaride alone allowed cells to progress through the cell cycle, but at a slower rate than control. Data from other labs have suggested that CaM inhibitors cause a G1/S block (Hidaka et al., 1981). It appears that zaldaride may cause cells to accumulate in S (note the higher percentage of cells in S compared to control (Table 1)), although there are still cells in G2/M. When Cd and zaldaride were combined, cells moved from G0/G1 into S, but still did not enter G2/M. This suggests again that zaldaride partially protects JAr cells from the effects of Cd. There is no complete protection, as cells did not progress into G2/M. This may be an indication that cells treated with Cd and zaldaride are able to complete the cell cycle, just at a slower rate, or that cells will now accumulate in S phase. The finding that, although Cd or zaldaride alone inhibited proliferation, Cd and zaldaride together protect against the inhibition of proliferation caused by Cd alone was unexpected. Cd binds to CaM, inducing a conformational change similar to calcium (Cheung, 1988). This chronic activation could disturb many cellular functions. This mechanism has been proposed in Cd-induced proximal tubule cell injury (Fowler et al., 1991). It has been postulated that Cd nephrotoxicity is attenuated by zinc induction of MT (Foster et al., 1991). CaM inhibitors (trifluoperazine, W7, calmidazolium, and chlorpromazine) have been shown to induce hepatic MT gene expression (Shiraishi and Waalkes, 1994). Trifluoperazine and W7 also increase MT protein levels (Shiraishi and Waalkes, 1994). The ability of zaldaride to induce MT has not been demonstrated, thus immunocytochemistry was performed to examine localization and abundance of MT protein. It was demonstrated that zaldaride alone does not induce MT protein levels when compared to Cd. In addition, MT protein in cells treated with both Cd and zaldaride does not differ from Cd alone, indicating that protection against Cd toxicity does not result from increased MT synthesis. The changes noted in MT localization in Cd-treated JAr cells corroborates data previously published by Breen et al. (1995). The effects of cadmium on hCG and progesterone synthesis were notably different. hCG production is initially decreased by Cd exposure, followed by a recovery which exceeds control production. Progesterone synthesis, on the other hand, is stimulated by Cd exposure throughout the culture period. It is not surprising to find a difference be-

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tween synthesis of a protein hormone (hCG) and a steroid hormone (progesterone), as they have completely different biosynthetic pathways. While zaldaride did not affect hCG and progesterone synthesis/release, synthesis/release of these hormones following the addition of zaldaride in combination with Cd did not differ from Cd treatment alone. Thus, although zaldaride reverses the toxicity of Cd on cell proliferation, it does not reverse the effects of Cd on protein and steroid hormone synthesis/release. While Cd alters both CT- and ST-like functions of JAr cells, there appears to be a cell-specific capability of zaldaride to reverse the effects of Cd. The primary target is the CT-like functions of JAr cells, with zaldaride affording protection against the inhibition of proliferation induced by Cd. hCG and progesterone syntheses/release, ST-like functions of JAr cells, appear to have a different response to the addition of zaldaride in conjunction with Cd. In this case, zaldaride does not reverse the effects of cadmium. This study demonstrates that placental toxicity induced by Cd can be mediated through inhibition of trophoblast cell proliferation. Inhibition of trophoblast cell proliferation by Cd can have a major impact on the establishment of a viable pregnancy. The addition of zaldaride to Cd reverses the inhibition of cell proliferation induced by Cd alone. Protection against Cd toxicity afforded by zaldaride does not appear to involve induction of MT, as treatment with zaldaride does not cause a change in expression and localization compared to controls. Thus, the mechanism of action by which Cd interferes with cell proliferation apparently involves the interaction of Cd with CaM. ACKNOWLEDGMENTS We thank Brenda Goodfriend and Regina Harley for their assistance in performing centrifugal elutriation and flow cytometry analyses. Dr. Christopher Cox and the Biostatistics Department of the University of Rochester were most helpful in performing statistical analyses. This work was supported in part by NIH Grants ES02774, ES0726, and ES01247 and the Mae Stone Goode Foundation. These data were presented in part at the 1996 Society of Toxicology meeting.

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