Simultaneous effect of lead and cadmium on granulosa cells: A cellular model for ovarian toxicity

Reproductive Toxicology 21 (2006) 179–185

Simultaneous effect of lead and cadmium on granulosa cells: A cellular model for ovarian toxicity Laxmipriya P. Nampoothiri a , Sarita Gupta b,∗ b

a Department of Biochemistry, Indian Institute of Sciences, Bangalore, India Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390002, India

Received 27 April 2005; received in revised form 6 July 2005; accepted 21 July 2005 Available online 12 September 2005

Abstract Lead (Pb) and cadmium (Cd) are known reproductive toxicants, which accumulate in granulosa cells of the ovary. Female Charles foster rats were treated with sodium acetate (control), lead acetate and cadmium acetate either alone or in combination at a dose 0.05 mg/kg body weight intra-peritoneally for 15 days daily. Animals were killed at proestrous stage and granulosa cells were isolated from the ovaries. Binding of 125 Iluteinizing hormone (125 I-LH), 125 I-follicle stimulating hormone (125 I-FSH) and 17␤-hydroxysteroid dehydrogenase activity were measured. As these receptors are localized on the surface of the cell membrane, we also estimated the membrane parameters of these cells. Our results demonstrated that both lead and cadmium caused a significant reduction in gonadotropin binding, which altered steroidogenic enzyme activity of granulosa cells. These changes exhibited a positive correlation with membrane changes of the granulosa cells. © 2005 Published by Elsevier Inc. Keywords: Lead; Cadmium; Ovary; Granulosa cells; Gonadotropin binding; 17␤-Hydroxysteroid dehydrogenase; Membrane parameters

1. Introduction Expanding urbanization and industrialization has led to an increase in environmental lead (Pb) and cadmium (Cd). A large body of literature indicates that these reproductive metal toxicants may accumulate in ovary and uterus [1,2] and cause cytotoxicity [3,4]. Two major cell types of the ovarian follicle, namely theca and granulosa cells, control ovarian functions by the production of various steroid hormones under regulation by the hypothalamic-pituitary axis. Studies on the steroidogenic capacities of isolated granulosa cells and theca cells led to the proposal of two-cell gonadotropin theory, which stated that theca cells produce C19 steroids which then diffuse into granulosa cell to produce estradiol [5]. This difference in steroid production is due to differential expression of key steroidogenic enzymes: 3␤-hydroxysteroid dehydrogenase (3␤ HSD) mainly expressed in theca cells; and 17␤-hydroxysteroid dehydrogenase (17␤ HSD) in the ∗

Corresponding author. Tel.: +91 265 2794544. E-mail address: [email protected] (S. Gupta).

0890-6238/$ – see front matter © 2005 Published by Elsevier Inc. doi:10.1016/j.reprotox.2005.07.010

granulosa cells which produces estradiol under the influence of follicle stimulating hormone (FSH) and luteinizing hormone (LH). In the adult, granulosa cells have both FSH and LH receptors that are maximally expressed during early and late proestrous stages of ovarian cycle, respectively [5]. Interference in receptor binding activities could reflect a change in female physiology due to environmental contaminants. Pb, for example, can affect the binding of FSH and LH in ovarian homogenates [6] and to decrease the gonadal steroid levels [3]. Cd is also known to decrease the serum gonadotropin [2] and gonadal steroid levels [7]. Much of the previous research on these metals has dealt with single metal exposure; however, a more realistic health scenario would likely include simultaneous effects of these metals in combination. Therefore, the net effect manifested by multiple exposure to Pb and Cd could be antagonistic, synergistic or additive. The objective of the present study was to examine the effects from simultaneous exposure of Pb and Cd on granulosa cells at exposure levels that cause minimum inhibition

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of steroidogenic enzymes without anticipated overt clinical indicators of toxicity. Previously, we demonstrated a decrease in serum gonadotropin levels [8] and serum progesterone levels [9] with simultaneous exposure to Pb and Cd. We also demonstrated that in vitro exposure of granulosa cells to Pb, Cd, or both in combination for 1 h causes a decrease in gonadotropin binding and steroid production [10]. The present study has investigated the in vivo effects of Pb and Cd either alone or in combination at the cellular level using a granulosa cell model. Parameters evaluated include gonadotropin binding and steroidogenic activity of granulosa cells. Since membrane integrity is important for gonadotropic binding, membrane parameters including cholesterol, phospholipids and fluidity were also evaluated. To our knowledge, there are no similar studies on simultaneous effects of Pb and Cd on hormone binding and membrane parameters in the ovarian granulosa system.

2. Materials and methods 2.1. Animals Animals were divided into four groups containing 12 animals each: Group I animals received sodium acetate and served as the control while Groups II–IV animals received lead acetate, cadmium acetate, or both in combination for 15 days [11]. The Pb and Cd dosing was 0.05 mg/kg body weight for animals treated with each alone. Animals in combined treatment group received 0.025 mg/kg Pb and 0.025 mg/kg Cd, for a total dose of 0.05 mg/kg [11]. Six animals from each group were killed at early proestrous stage for FSH binding and at late proestrous stage for LH binding. Animals were synchronized such that they exist in proestrous stage after 15 days of metal exposure. After the experimental regime, rats were killed by decapitation; blood were collected and after digestion with acid, used for metal estimation. Serum was separated from aliquot of blood and was stored at −20 ◦ C for the assay of serum hormones.

2.3. Metal analysis Pb and Cd concentrations were determined in ovary. The samples were digested in reagent grade nitric acid–perchloric acid (2:1) mixture. The digestion was continued until samples became colorless. Then, the acid mixture was evaporated and the residue was dissolved in few drops of concentrated HCl. The sample was diluted to 1 ml with distilled water and the readings were taken in GBC 902 double beam atomic absorption spectrophotometer. Sensitivities of the assays were 0.06 and 0.009 ␮g/ml for lead and cadmium, respectively. Detection limit using the instrument were 0.05 ng/ml for lead and 0.003 ng/ml for cadmium, respectively. 2.4. Granulosa cell isolation Ovaries were removed and granulosa cells were isolated by a modification of the method used by Campbell [12]. In brief, ovaries were suspended in Hanks balance buffered saline (HBSS) and treated with hypertonic sucrose and EGTA solutions. The ovaries were expressed with blunt spatula and resuspended in HBSS. Cell viability was checked by trypan blue staining. Purity of granulosa cells was evaluated by assessing 3␤-hydroxysteroid dehydrogenase activity, which is not detected if the preparation of cells is pure. 2.5.

125 I-LH

and 125 I-FSH binding

Rat LH and FSH were iodinated by the method of Chloramine-T method [13]. Radioreceptor assay was performed according to the method of Guerrero et al. [14]. About 100,000 live cells were taken and then incubated with labeled hormone for 24 h at 4 ◦ C. After incubation, the cells were rinsed twice with phosphate buffered saline (PBS) and centrifuged at 3000 rpm for 10 min. Radioactivity in the cell pellet was measured in gamma counter. Nonspecific counting was determined by incubating the cells with 125 I-LH or 125 I-FSH, in presence of excess unlabelled r-LH (100 ␮g) and unlabelled r-FSH (1000 ng). Binding of labeled gonadotropins was represented as number of receptors present on the ovary.

2.2. Chemicals 2.6. Assay of steroidogenic enzymes Dihydroepiandrostenedione (DHEA), 17␤-estradiol, bovine serum albumin (BSA). Ethylene glycol tetra acetic acid (EGTA), Chloramine-T and Oubain were purchased from Sigma Inc., USA. Indole nitro tetrazolium (INT), Tween-20, NAD were purchased from Sisco Research laboratories Ltd., Mumbai, India. Trypan blue was obtained from Hi-Media Labs, India. Radio Immuno Assay Kits were procured from Immunotech, Germany. Na-I125 (2 mCi) was purchased from BRIT, India. Rat luteinzing hormone (AFP 115368) and rat follice stimulating hormone (AFP 19896) were obtained from National Health Pituitary Program (NHPP), NIDDK, Bethesda, USA as a kind gift from Dr. Parlow.

Enzymatic activity of 17␤ HSD was estimated by the method of Shivanandappa and Venkatesh [15]. The assay system contains 0.1 M Tris–HCl (pH 7.8), 5 mM NAD, 1 mM DHEA/17␤-estradiol, 0.4 ␮M INT and the 4 × 105 cells in a total volume of 3 ml, which was incubated for 1 h at 37 ◦ C. The reaction was terminated using 50 mM potassium phthalate buffer and absorbance was measured at 490 nm. 2.7. Preparation of granulosa cell membrane Membrane was prepared according to the protocol of Riordan and Ling [16]. About 10,000 live granulosa cells were

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taken and sonicated at five cycles for 2 min in HBSS. After ultrasonic disruption the cells were spun at 1000 × g for 10 min and supernatant was centrifuged at 20,000 × g for 1 h twice. The pellet obtained contained the purified membrane fraction, which was resuspended in 10 mM Tris.

incubation medium; the Na+ K+ ATPase activity was calculated as the difference between the total and Mg2+ ATPase activity.

2.8. Measurement of membrane cholesterol

Results were expressed as mean ± standard error. All data were analyzed employing two-way ANOVA followed by Bonferroni post test for multiple comparisons. Values of p < 0.05 were considered of statistical significance.

Membrane cholesterol was estimated by the method of Leffler and McDougald [17]. The membrane samples were treated with 1 ml of Ferric chloride-acetic acid solution (1 mg/1 ml) in presence of 2 ml of sulfuric acid to give a colored product whose absorbance was measured at 540 nm.

2.12. Statistical analysis

3. Results 3.1. Metal analysis in the ovary

2.9. Measurement of membrane phospholipids We measured the phospholipid content of granulosa cell membrane by the extracting the phospholipids by using the protocol of Folch et al. [18]. The extracted lipids were digested with acid mixture (1:1:2 ratio of perchloric acid: sulfuric acid: distilled water), which was followed by estimation of phosphorus by Barlett method [19].

There were no significant differences in body weight in treated animals as compared to control animals. The concentrations of Pb and Cd in the ovary after 15 days of exposure are represented in Table 1. Both metals were accumulated in the ovary after exposure. The animals receiving combined treatment exhibited intermediate values for both lead and cadmium metals compared to individual metal treated groups.

2.10. Measurement of membrane fluidity

3.2. Effect of metals on granulosa cell count

Fluorescence polarization studies were performed as indicated by Shinitzky and Barenholz [20]. A suspension of granulose cell membrane was incubated for 1 h with 0.6 ␮mol/L 1,5-diphenyl-1,3,5-hexatriene (DPH) in 0.32 M sucrose solution containing 10 mM Tris–HCl, pH 7.4. The excitation and emission wavelengths were 360 and 430 nm, respectively, with bandwidths 5 and 10 nm, respectively. Polarization values for parallel (vertical) and perpendicular (horizontal) were taken with a Shimadzu RF-540 Spectroflurometer. The polarization value (P) was calculated from the equation

Animals treated with Pb exhibited a maximum decrease in their cell count compared with other metal treated groups. Cells of combined treated group demonstrated an intermediate decrease in granulosa cell number compared to control (Table 2).

IVV − GIVH P= IVV + GIVH where Ivv and IVH are vertical and horizontal components of emitted light, respectively, when emitted with vertically polarized light, and G is the correction factor for emission monochromator.

Table 1 Effect of lead and cadmium alone and in combination on ovarian lead and cadmium content (ng/mg wet weight), at a dose of 0.5 mg/kg body weight daily for 15 days Groups

Lead

Cadmium

Control Lead Cadmium Lead + cadmium

N.D 3.14 ± 0.24a N.D. 1.53 ± 0.26a,b

N.D N.D 3.52 ± 0.01a,b 2.27 ± 0.03a,c,b

N.D. = not detectable (N = 3–4). The values are mean ± S.E.M. a p < 0.001 compared to control. b p < 0.001 compared to lead group. c p < 0.01 compared to cadmium group.

2.11. Na+ K+ ATPase assay ATPase activity was measured by the release of inorganic phosphorus from ATP [21]. The Pi was assayed according to Fiske and Subbarow [22]. The incubation mixture contained 50 mmol/l Tris–HCl (pH 7.4), 100 mmol/l NaCl, 20 mmol/l KCl, 3 mmol/l MgCl2 , 3 mmol/l ATP, and 100 ␮g membrane protein, in a final volume of 1 ml. The assay was initiated with ATP. Enzyme incubation lasted for 20 min at 37 ◦ C in a water bath, and the reaction was stopped by addition of cold trichloroacetic acid to 10% (v/v). To determine the basal Mg2+ ATPase activity, we added 0.2 mmol/l oubain to the

Table 2 Effect of lead and cadmium either alone or in combination on granulosa cell count at a dose of 0.5 mg/kg body weight daily for 15 days Groups

Number of cells × 106 per mg ovary

Control (NaAc) Lead Cadmium Lead + cadmium

0.028 0.017 0.0228 0.0241

N = 5–6. The values are mean ± S.E.M. a p < 0.01 compared to control. b p < 0.05 compared to control.

± ± ± ±

0.045 0.035a 0.041b 0.045

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Fig. 1. Effect of lead and cadmium either alone or in combination on gonadotropin binding on rat granulosa cells (N = 6). The values are mean ± S.E.M. a p < 0.0001, b p < 0.0003 and c p < 0.001 compared to control. 1 p < 0.0001, 2 p < 0.0002 and 3 p < 0.02 compared to lead group. * p < 0.0001 and ** p < 0.0002 compared to combined treated group.

Fig. 3. Effect of lead and cadmium either alone or in combination on membrane parameters of granulosa cells (N = 5–6). The values are mean ± S.E.M. a p < 0.0002, z p < 0.0006, b p < 0.0017 and c p < 0.005 compared to control. 1 p < 0.0002, 2 p < 0.0009 and 3 p < 0.001compared to lead group.* p < 0.02 and ** p < 0.004 compared to combined treated group.

3.3. Effect of metals on gonadotropin binding Binding of 125 I-rFSH and 125 I-rLH to granulosa cells in various groups are shown in Fig. 1. Cells of Cd-treated animals demonstrated a maximum decrease in binding of both gonadotropins (LH and FSH) as compared to combined treatment; while lead treated cells showed minimum inhibition in binding as compared to cells of control group. 3.4. Effect of metals on steroid dehydrogenase activity Analysis of 17␤ HSD activity in granulosa cells of ovary showed that cells of Cd treated group showed maximum inhibition (55.5%, p < 0.001) while cells of the combined treated group showed intermediate results with 49% inhibition (p < 0.001). Cells of the Pb-treated group demonstrated a minimal inhibition (43.2%, p < 0.001) as compared to cells of the control group (Fig. 2). 3.5. Effect of metals on membrane parameters Both cholesterol and total phospholipid contents were maximally decreased in the granulosa cell membrane of Cdexposed animals, while both the components demonstrated a minimum decrease in Pb-treated groups (Fig. 3). Membrane

Fig. 2. Effect of lead and cadmium alone and in combination on 17␤hydroxy steroid dehydrogenase activity in granulosa cells (N = 6). The values are expresses as mean ± S.E.M. a p < 0.0001 and b p < 0.005 compared to control group. 1 p < 0.0007 compared to lead group. * p < 0.02 compared to combined treated group.

Fig. 4. Effect of lead and cadmium either alone or in combination on granulosa cell membrane fluidity (N = 4–5). The values are mean ± S.E.M. a p < 0.0001, b p < 0.0011 and c p < 0.02 compared to control group. 1 p < 0.0001 and 2 p < 0.0004 compared to lead group. * p < 0.0007 compared to combined group.

fluidity was increased maximally in membrane of cadmiumexposed animals compared to control. The membrane of granulosa cells of Pb-treated animals exhibited a minimal change in both fluidity and cholesterol to total phospholipid ratios. Combined treated animals showed an intermediate change in fluidity (Fig. 4). 3.6. Effect of metals on Na+ K+ ATPase Granulosa cell membrane of cadmium treated animals exhibited a maximum inhibition in the activity of Na+ K+ ATPase while exposure to lead demonstrated a minimal inhibition as compared to control. The exposure of animals to combined metal showed an intermediate value (Fig. 5).

Fig. 5. Effect of combined exposure of lead and cadmium on granulosa cell membrane Na+ K+ ATPase activity (N = 5–6). The values are mean ± S.E.M. a p < 0.0001 and b p < 0.0016 compared to control. 1 p < 0.0009 and 2 p < 0.009 compared to lead group. * p < 0.02 compared to combined treated group.

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4. Discussion To our knowledge this is the first study to demonstrate the effects of both Pb and Cd in combination on gonadotropin binding and their effect on steroidogenesis, in correlation with cell membrane changes. The ovary is vital for female steroid hormone synthesis. Theca and granulosa cells in response to these steroids control estrus cyclicity. Effects of potential reproductive toxicants such as Pb and Cd on female physiology is difficult to interpret due to differences in sensitivity of metals, concentration of metals present in the tissue, and time-duration of exposure [23]. Our findings demonstrated the pattern of heavy metal accumulation in ovary. The combined (Pb + Cd) treatment group exhibited an intermediate concentration of the toxicants when compared to the individual metal treated groups. This could be due to the competition between the metals for uptake by cells in the ovary. We also have reported earlier that metal exposure caused accumulation of metals in other organs such as liver [11] and pituitary [8]. The accumulation could be due to apparent deficiency of metallothioneins [24]. Paksy et al. [3,4] reported that Pb and Cd are concentrated in the granulosa cells. The decrease in cell count demonstrated in the present study is likely the result of cytotoxic effect of metal toxicants. In this study, Pb appeared more cytotoxic than Cd as suggested by histological findings (data not shown). In other studies of the ovary in animals exposed to Pb, rats were observed to develop ovarian cysts at high exposure levels [25]; female mice had reductions in small and medium follicle numbers [26]; and dysfunction of folliculogenesis correlated with fewer primodial follicles and atretic antral follicles [27]. All these data imply that Pb intoxicaion alters folliculogenesis and granulosa cell counts in mammals. We previously reported the decrease in ovarian weight along with decrease in cell count on combined exposure to Pb and Cd [9]. The gonadotropins LH and FSH bind specific receptors on the granulosa cell surface to stimulate second messenger systems and gonadal steroid production. The concentration of Pb or Cd present in the tissue, and the time of exposure to the toxicants [28], could affect hormone receptor kinetics, enzyme activities and/or hormone secretion. Using a granulosa cell model to understand the mechanism of toxicants, the our results demonstrated that Cd treatment caused a maximum decrease in binding of peptide hormones (both LH and FSH) as compared to the combined treatment group, whereas Pb treatment had minimum inhibition versus the control group. Wiebe et al. [6] also reported a decrease in binding of LH and FSH in ovarian homogenate. Several reports demonstrated that LDL receptor and cytochrome P450 systems are affected by Pb and Cd [29–31]. The present study clearly demonstrated decreased receptor binding, which is important for decreased steroidogenesis since it is well known that gonadotropins via binding to specific cell surface receptors regulate cholesterol uptake as well as the rate-limiting steps in steroidogenesis, suggesting the importance of LH and FSH

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receptors. Recently, we reported the decreased binding of gonadotropins in granulosa cells on in vitro metal exposure [10]. The percent decrease obtained with in vivo metal exposure was high as compared to the in vitro exposure scenario, suggesting the importance of time of exposure and role of other factors in metal toxicity. Gonadotropin receptors are membrane bound receptors. Membrane integrity is based on the lipid bilayer, which provides fluid state for the structural organization of membrane proteins. It is known that heavy metals can alter membrane integrity [32,33]. This alteration could affect receptor confirmation and thus contribute to decreased binding of gonadotropins. Our results have showed a decrease in total cholesterol and phospholipid content, along with an increase in fluidity of granulosa cell membrane, in all exposed groups. Amoruso et al. [34] also reported an increase in fluidity of erythrocyte membrane on exposure to Pb. The change in membrane fluidity could be due to oxidation of double bonds of fatty acids, formation of lipid peroxides and interaction of heavy metals with negatively charged groups of phospholipid as suggested by Fiorini et al. [35]. Several authors reported that heavy metals could generate free radicals, which in turn alter membrane proteins including Na+ K+ ATPase [36,37]. The present study demonstrated an inhibition of Na+ K+ ATPase activity in all the metal treated groups in granulosa membrane. Indeed, earlier reports have also indicated that Pb inhibits K+ dephosphorylation step of Na+ K+ ATPase [38,39] and Cd interacts with enzyme–phosphate complex [40], thus inhibiting the enzyme activity directly apart from the fluidity change. Taken together, the evidence is consistent with a role for membrane integrity in decreased gonadotropin binding as observed here; however, the possibility of other factors interacting with heavy metals cannot be ruled out. Membrane receptors may complex with cytoskeletal elements to influence their mobility [41], and Pb and Cd are known to cause disassembly of cytoplasmic microtubule complex and change receptor mobility [42,43]. Furthermore, Pb and Cd may interact with cystine residues of the receptor protein all contributing to the stability of hormone–receptor complex structure and binding affinity. 17␤ HSD is a marker enzyme for granulosa cells [44] belonging to the class of short chain dehydrogenases [45]. Our study demonstrates that metal salt caused a significant decrease in this enzymatic activity. The decrease could be attributed to indirect mechanisms such as reduced gonadotropin binding as well as direct interaction of metal with the amino acids present on the active site of the enzyme or to –SH groups of cysteine residue present at the NAD binding domain [45]. Paksy et al. [46] reported that Cd enters the granulosa cell and causes a dose dependent decrease in estradiol production, which could be an effect mediated by interference of Cd with the aromatase system. Taupeau et al. [47] reported that in vitro exposure of Pb (as Pb acetate) to human granulosa cells caused a decrease in basal activity of aromatase enzyme, along with reduced mRNA levels. Inhi-

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bition of hormone synthesis may partly involve the ability of Cd and Pb to compete with calcium [48,49]. Veldhuis et al. [50] reported that a calcium-deficient medium impedes the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous substrate, thereby indicating a plausible role of calcium in steroidogenesis. Calcium-calmodulin systems are known to play a fundamental role in follicular steroidogenesis [51]. Furthermore, it has been demonstrated that cadmium and lead can bind with calcium binding site’s of calmodulin and disorderly regulate calcium-calmodulin dependent functions [52,53]. These findings suggest there exists competition between the metals to bind to the same site. It is to be noted that combined exposure of Pb and Cd revealed intermediate results among the various parameters. When both metals are present in similar concentrations (as in present study) Cd may dominate due to its more reactive nature. Also, the intermediate effects seen in most parameters studied upon combined exposure to both toxicants in similar concentration is related to competition between the two metals to exhibit their effect. Thus, the adverse effect on peptide hormone binding by the simultaneous exposure of toxicants is correlated with decrease in steroid dehydrogenase activity as well as alteration in lipid bilayer. The present study offers preliminary findings to improve understanding of the biochemical mechanism of metal toxicity at the cellular level. Additional parameters such as cell signaling must be analyzed to further understand other biochemical and molecular mechanism of metal interaction. This study accounts for altered ovarian and reproductive function due to lead and cadmium exposure as reported by several workers. Acknowledgements The authors are thankful to NIDDK National Hormone and pituitary Program (NHPP, USA) and Dr. Parlow for the gift of LH and FSH. Thanks are due to Prof. Michael Aruldas and Mr. Venkatesh of P.G. Institute of Basic medical Sciences, University of Madras, Chennai, India for helping in performing the assays. This study has a financial aid from CSIR grant no. 9/114(103)/98/EMR-I.

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