Estrogenic and toxic effects of polychlorinated biphenyls on cultured ovarian germ cells of embryonic chickens

Reproductive Toxicology 19 (2004) 79–86

Estrogenic and toxic effects of polychlorinated biphenyls on cultured ovarian germ cells of embryonic chickens Meina Xie, Caiqiao Zhang∗ Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, No. 268 Kaixuan Road, Hangzhou 310029, PR China Received 5 February 2004; received in revised form 1 June 2004; accepted 4 June 2004 Available online 20 July 2004

Abstract Polychlorinated biphenyls (PCBs) are man-made ubiquitous pollutants that have detrimental effects on reproduction and endocrine functions in a variety of species. In the present study, estrogenic and toxic effects of PCBs on embryonic chicken ovarian development were evaluated by a germ-somatic cell co-culture system. Ovarian cells were cultured in serum-free medium and challenged with a mixture of PCBs (Aroclor 1254). Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release. Results showed that lower PCBs (0.1–1 ␮g/ml) manifested mainly estrogenic effect to stimulate germ cell proliferation, while higher PCBs (10 ␮g/ml) imposed severe toxicity on germ and somatic cells. The toxic effect of PCBs could be attenuated by an antioxidant tocopherol. PCBs induced condensed nuclear chromosome in ovarian cells and caused cell exfoliation and breakdown within initial hours of treatment. After 24 h, the estrogenic effect of PCBs began to exhibit and the survived germ cells manifested proliferation. Inhibition of the estrogenic effect of PCBs by tamoxifen led to increased toxicity on germ cells and somatic cells. These results indicate that PCBs exposure may interfere with ovarian germ cell proliferation and cause reproductive disorder via both toxic and estrogenic actions in embryonic chickens. © 2004 Elsevier Inc. All rights reserved. Keywords: Polychlorinated biphenyls; Chicken; Ovary; Germ cell; Estrogen

1. Introduction During the past 50 years, a variety of synthetic chemicals and their by-products have been released to the environment as a consequence of efforts to increase agricultural productivity or modern manufacturing, which caused new problems for environment. In recent years, much attention has focused on the potential for a wide range of xenobiotic chemicals to interact with and disrupt the endocrine systems of animal and human populations. As common environmental pollutants, PCBs include 209 possible congeners with a variety of chlorine substitution patterns. PCBs were synthesized for approximately 60 years from 1929 until they were restricted in many countries during the late 1970s; however, they were produced in Russia until 1999 and continue to be used in most of the world even today. Their uses varied from closed-system applications in capacitors and trans-

∗ Corresponding author. Tel.: +86 571 869 71976; fax: +86 571 869 71976. E-mail address: [email protected] (C. Zhang).

0890-6238/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2004.06.004

formers to open-system application in the manufacture of adhesives, textiles printing and cooling systems etc. Obviously, such a plethora of uses has facilitated their ubiquity in the environment. Because of their resistance toward biotransformation, PCBs accumulate in the food chain and can be found at all levels of the food chain. PCBs have been identified in practically all the environmental matrices and in human tissues including adipose tissue, blood and milk [1–3]. Although PCBs are not necessarily fatal to wildlife and human, their detrimental effects can result in dramatic decline or complete loss of populations due to their ubiquitous, persistent and lipophilic characters. Many of these compounds can mimic sex steroid activities; therefore, they are potential endocrine disrupters causing increased risk of reproductive disorders and carcinogenesis. Many studies showed that exposure to PCBs in early life induced developmental abnormities. Reproductive abnormalities caused by these environmental pollutants were observed in wildlife and experimental animals, including inhibition or destruction of spermatogenesis in cods and cocks [4,5], degeneration of rabbit preimplantation embryos and rat seminiferous

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tubule, epididymal and testicular lesion [6]. Estrogenic activity associated with PCBs in an uterotrophic model was reported 30 years ago. Subsequently, several studies had reported that commercial PCB mixture or individual congeners and/or their hydroxylated metabolites exhibited estrogenic or anti-estrogenic activity [7,8]. PCBs have been detected in human ovarian tissue, embryos and fetuses [9]. Rodents exposed to PCBs had experienced a reduction in germ cell number [10]. Some of these studies suggested direct and indirect effects of PCBs on ovarian function. For example, rats exposed to the commercial mixture Aroclor 1242 caused a reduction in the number of follicles [11]. Kholkute et al. revealed toxic effects of technical mixtures, such as Aroclor 1254 and 1268, on in vitro maturation and fertilization of mouse oocytes [12]. Data about the direct effects of PCBs on germ cells are still scarce and no studies have been reported about the direct effects of PCBs on cultured germ cells. Aroclor 1254 is a technical mixture of PCBs whose composition is considered as environmentally relevant at a certain extent [13]. Mixtures of PCBs are more toxic than individual congeners [14]. This is of ecological significance since mixtures, not individual congeners, were used industrially and have accumulated in the environment [15,16]. Our previous in vivo studies showed that the adverse effects of Aroclor 1254 on early chicken embryonic gonadal and germ cell development were initiated during the early embryonic stage [17]. Since avian embryos, unlike mammalian embryos, develop independently of maternal environment during incubation, so it is much easier to determine the in vivo effects of exogenous chemicals without maternal interference. In the present study, an in vitro germ-somatic cell co-culture model was established to reveal the direct effects of PCBs on ovarian cells. In this co-culture system, a monolayer of somatic cells with attached germ cells was convenient for assessment of morphological changes of both germ and somatic cells. Cytotoxicity of ovarian cells was determined by MTT reduction and LDH leakage assay. In addition, an estrogen receptor antagonist tamoxifen and an antioxidant, tocopherol (vitamin E), were used to distinguish the mechanism of toxic effect and estrogenic effect of PCBs on ovarian cells.

2. Materials and methods 2.1. Animals Fertilized Avian chicken eggs were obtained from a local hatchery and incubated at 38.5 ◦ C and 60% humidity until 18-day-old. 2.2. Culture of ovarian cells Left ovaries were removed from day 18 chicken embryos under sterile conditions. Ovaries were minced into small

fragments (about 1 mm3 ) and were digested in 1 mg/ml collagenase (GIBCO BRL) at 37 ◦ C in a shaking water bath. Two-step digestion (15 min for the first time and 10 min for the second time) was performed to obtain more cells. The dissociated cell aggregates were collected and filtered through a 150 ␮m mesh, and then washed with fresh McCoy’s 5A medium (GIBCO BRL) three times by centrifugation at 1000 rpm for 10 min. Cell viability was greater than 90% evaluated by a trypan blue exclusion test. The cell suspension was added to collagen-treated 96-well flat-bottom culture plates (Nunc, Denmark) at a density of 1 × 105 /well (for MTT and LDH assay) or 5 × 104 /well (for morphological studies) in 200 ␮l serum-free medium and cultured at 39 ◦ C in a humidified atmosphere of 5% CO2 . The basal medium was supplemented with 200 mM glutamine, 10 ␮g/ml insulin, 5 ␮g/ml transferrin and 3 × 10−8 M selenite (Sigma) as ITS medium. 2.3. Treatment of cultured cells At the same time of culture, cells were challenged with PCBs and other chemicals. A mixture of PCBs, Aroclor 1254 (99.99% pure, lot 124-191-A, Accustandard Inc., New Haven, Connecticut, USA) was dissolved in ethanol as a stock solution and diluted in medium to different concentrations (0.1, 1, 10 ␮g/ml) for treatment of cells. 17␤-estradiol (E2 ), tocopherol and estrogen receptor antagonist tamoxifen (Sigma) were also dissolved in ethanol and diluted in medium. The cultured cells received E2 (1 ␮g/ml), tocopherol (10 ␮g/ml), or tamoxifen (1 ␮g/ml) alone or in combinations with PCBs (10 ␮g/ml). The final concentration of ethanol in the medium was below 0.2%. The control received vehicle only. 2.4. Morphological studies and count of ovarian germ cells Proliferation and morphology changes of germ and somatic cells were observed under an inverted phase-contrast microscope (IX70, Olympus). Photos were transferred into computer by a digital camera (Pixera Pro 150ES). The numbers of germ cells culture for 48 h were counted and analyzed by using SimplePCI Advanced Imaging Software (Compix Inc.). 2.5. Cell proliferation test by MTT reduction The cytotoxicity on cell proliferation was quantified by measurement of the reduction of MTT to produce a dark blue formazan product. After incubation for 6 h with treated chemicals at the indicated concentrations, MTT (Sigma) was added to each well at a final concentration of 0.25 mg/ml and the plate was incubated at 39 ◦ C in a CO2 incubator. After 4 h incubation, the medium was removed, and 100 ␮l DMSO was added to each well to dissolve the formazan crystals and the optical density (OD) was measured at a

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Fig. 1. Morphological changes of embryonic chicken ovarian cells after treatment with Aroclor 1254 and Tocopherol in serum-free ITS-supplemented McCoy’s 5A medium for 6 h. A, B and C indicates ovarian cells of control, Aroclor 1254 (10 ␮g/ml) and Aroclor 1254 (10 ␮g/ml) + Tocopherol (10 ␮g/ml)-treated group, respectively. Somatic cells (arrowhead) began to expand as shuttle-shape, large round germ cells (arrow) grow upon somatic cells in A. Condensed nuclei and cytoplasm vacuole occurred in germ cells and many cell pieces were released into the medium in B. While in C, cell damage was attenuated. Bar indicates 10 ␮m (magnification 400×).

wavelength of 570 nm using a microplate reader (Multiskan MK3).

3. Results 3.1. Morphology of ovarian cells in culture

2.6. LDH release assay Ovarian cells injury was quantitatively assessed by the measurement of LDH released from damaged or destroyed cells in the extracellular fluid 6 h after treatment by a LDH kit (Nanjing Jiancheng Bioengineering Institute). LDH activity is proportional to the rate of pyruvate loss, which was assayed by absorbance change at a wavelength of 440 nm.

2.7. Statistics All experiments were performed in four replicates for each concentration and treatment group. The experiments were repeated three times. Data were expressed as the means ± S.D. and evaluated by analysis of variance followed by SAS 6.12 test. P < 0.05 was considered significantly different.

The cell suspension obtained from the whole ovary contained germ cells (mainly were oogonia and primary oocytes) and somatic cells (granulosa, interstitial and epithelial cells etc.). The diameter of germ cells was between 15 and 25 ␮m, greater than somatic cells (arrow, Fig. 1A). After culture for 24 h in serum-free ITS medium, somatic cells (arrowhead, Fig. 3A) had attached at the bottom of the culture plate, growing as shuttle-shape and almost spread around the whole bottom to form a monolayer at 48 h of culture. Germ cells (arrow, Fig. 3A) as round or oval shapes were attached in the free surface of the aggregate of somatic cells. In this co-culture system, germ cells could survive and kept proliferating for at least 10 days in ITS medium. 3.2. Assessments of ovarian cells damage by PCBs After culture for 1 h, cytolysis and disorganization of cells were found in the group of 10 ␮g/ml PCBs. After treatment

Fig. 2. Assessments of ovarian cells proliferation and cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) reduction (A) and LDH (lactate dehydrogenase) leakage (B) after treatment for 6 h. Each bar represents the mean ± S.D. of four determinations. Bars with different superscripts were statistically different (P < 0.05, n = 4).

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Fig. 3. Morphological changes of embryonic chicken ovarian cells after treatment in serum-free medium for 48 h. A, B, C, and D indicated ovarian cells of control, Aroclor 1254 (1 ␮g/ml), Aroclor 1254 (10 ␮g/ml) and E2 (1 ␮g/ml)-treated group, respectively. More germ cells (arrows) appeared above the somatic cells (arrowhead) in B, C, and D than in A. There were also toxic effects of Aroclor 1254 (10 ␮g/ml) on cells (C). Bar indicates 10 ␮m (magnification 400×).

for 6 h, the damaging effects of PCBs were very obvious. Condensed nuclei and cytoplasm vacuole occurred and many cell pieces were released into the medium (Fig. 1B). While in the PCBs plus tocopherol group, cytotoxicity of PCBs was attenuated (Fig. 1C). Significant losses in cell viability occurred in ovarian cells exposed to PCBs at 10 ␮g/ml after 6 h of incubation. MTT assay showed significant reduction in remaining alive ovarian cells over 6 h treatment with PCBs (10 ␮g/ml) (P < 0.05) (Fig. 2A). Tocopherol could decrease the mortality of ovarian cells and the OD value was higher than that of PCBs (10 ␮g/ml) treated only (P < 0.05) (Fig. 2A). There was no obvious difference between lower PCBs groups (0.1–1 ␮g/ml) and control. Ovarian cells damage was also assayed by LDH release (Fig. 2B). In all groups the visual estimate was consistent with the results obtained with the MTT assay. There were significant elevations (P < 0.05) in LDH leakage from ovarian cells after treatment with 10 ␮g/ml PCBs for 6 h. The toxic effect of PCB on LDH leakage was partly inhibited by tocopherol co-treatment (P < 0.05).

(0.1–1 ␮g/ml) (P < 0.05) (Figs. 3B and 4). Compared with the control group, germ cells in the highest PCBs (10 ␮g/ml) group also showed an increase (P < 0.05) (Figs. 3C and 4). Meanwhile, toxic effects of PCBs on germ and somatic cells were still retained, especial on somatic cells that displayed decreased expansion at the bottom of culture plate (Fig. 3C). Tocopherol could antagonize the toxic effects of PCBs (Fig. 5B). As a positive control, the stimulatory of germ cell number was found in E2 -treated group (Figs. 3D

3.3. Changes in ovarian germ cell number

Fig. 4. Effects of Aroclor 1254 on germ cell proliferation of embryonic chicken ovary after 48 h in serum-free medium. Germ cells number increased significantly in all PCB-treated groups (P < 0.05, n = 4). Values are the mean ± S.D. Bars with different superscripts were statistically different (P < 0.05, n = 4).

After culture for 48 h, the number of germ cells displayed a significant increment stimulated by lower PCBs

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Fig. 5. Morphological changes of ovarian cells by treatments of tocopherol and tamoxifen alone and in combinations with Aroclor 1254 for 48 h. A, B, C, and D indicated ovarian cells treated with tocopherol (10 ␮g/ml), Aroclor 1254 (10 ␮g/ml) + tocopherol (10 ␮g/ml), tamoxifen (1 ␮g/ml), Aroclor 1254 (10 ␮g/ml) + tamoxifen (1 ␮g/ml), respectively. Germ cell number in B was much more than A and cell damage was attenuated in B compared with that in PCBs-treatment alone. In combination with tamoxifen, PCBs manifested severer toxic effect on cell morphology and proliferation (D) compared with that in PCBs-treatment alone. Bar indicates 10 ␮m (magnification 400×).

and 4). In combination with tamoxifen, PCBs manifested increased toxic effect on germ cell morphology and proliferation compared with PCBs-treatment alone (Figs. 4 and 5D).

4. Discussion Embryonic exposure to environmental contaminants served as a model for assessing the potential toxicity of a broad range of chemicals with estrogenic activity. The embryo toxicity to birds had been primarily studied by direct injection of PCB congeners or commercial mixtures into egg (air cell or yolk sac), and then monitoring embryo mortality and modality changes in organ development and functions, especially those related with reproduction [17,18]. The differentiation and development of the gonads are under complex regulation of hormones and growth factors. Since estrogen plays a key role in gonadal differentiation in the course of embryo development, environmental endocrine disrupters may impose greater negative effects on gonadal development at this sensitive stage. In vitro culture is becoming increasingly common in evaluating the effects of drugs, microorganisms or their products on reproductive performance and this provides further help for clinical ap-

plications. A germ-somatic cell co-culture model was established in the present study to evaluate the effects of Aroclor 1254 on proliferation of ovarian germ and somatic cells of chicken embryos in vitro. In this co-culture system, germ cells developed well for a long period over 10 days and could be observed conveniently as the somatic cells served a monolayer in serum-free medium without overgrowth. Moreover, this system is sensitive to the endogenous hormones such as follicle-stimulating hormone and exogenous toxicants. Therefore, it can be used as a model for evaluating actions of hormones and drugs, cytotoxicity and genotoxicity by reproductive toxicants in vitro. With this co-culture model we observed stimulated proliferation of germ cells by E2 , and damage of germ cells by PCBs. The morphological changes of germ cells could be easily monitored to evaluate effects of hormones or endocrine disrupters. PCBs have been detected as contaminants in almost every component of the global ecosystem including air, water, soil, wildlife, and human adipose tissue, milk and serum [19]. Human is at the top of the food chain and will, therefore, contain relative high amounts of xenobiotics. Most studies had focused on fetal toxicity, developmental malformation, decrease in reproductive ability, and cancers [20,21]. For example, exposure of rats to PCBs in utero during critical period of development led to reduced number of preantral and

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antral follicles of certain size classes [22]. Our in vivo studies also revealed the estrogenic effect of PCBs on ovarian cortex development and this effect could be blocked by an estrogen receptor antagonist clomiphene [23]. Estrogenic or antiestrogenic activity was reported for both PCBs and hydroxylated PCBs [24,25]. Estrogens at concentrations above physiological level were antagonists of the androgen receptor (AR) [26], so the estrogenic or antiestrogenic activities of PCBs possible rely on different levels. In the germ-somatic cell co-culture, treatment of PCBs at 0.1/1 ␮g/ml promoted proliferation of ovarian germ cells, which mimicked the action of endogenous estrogen. At 10 ␮g/ml, PCBs manifested intensive toxic effect on germ cells and somatic cells besides estrogenic effect. These in vitro effects of PCBs resembled the in vivo studies on chicken embryos in which PCBs exhibited significant increment in ovarian transverse section areas, thickness of the ovarian cortex and the total number of oocytes. Some oocytes showed vacuolated cytoplasm and hyperchromatic nucleus [17,23]. As the level (0.1–10 ␮g/ml) we chose in this experiment could also be found in some birds [27–29], PCBs and related contaminants were regarded as one reason for the decline of reproductive functions in wild animals. Some PCBs had been shown to exert potential estrogen and antiestrogen effects mediated via aryl hydrocarbon receptor (AHR) which included downregulation of the estrogen receptor (ER), interference of ligand activated ER binding to DNA response element and/or induction of cytochrome P450. All of these steps were involved in metabolism of estradiol [30,31]. PCBs capable of eliciting estrogenic activity were also hypothesized as a contributing factor in the development of hormone-related cancers and disrupting effects on reproduction in humans and wildlife [32]. As estrogen receptor antagonist could attenuate the estrogenic effect of PCBs, PCBs may elicit its hormonal action at least partly via the same receptor as endogenous estrogen. The detrimental effects of PCBs on female reproduction system had been focused in recent years. In the present study, the acute effect of PCBs exhibited directly severe damage on germ cells and somatic cells. The toxicity appeared after exposure to PCBs for 1 h and intensified after 6 h in both germ and somatic cells, with condensed cytoplasm and splintered membrane. Many cells fell off and then broke into pieces. This toxic effect of PCBs was similar to that observed in the in vivo studies. Injection of PCBs (1–100 ␮g/egg) into chicken egg yolk during incubation resulted in a dose-independent increase in mortality, cytoplasm vacuolation and nuclear hyperchromatism in primordial germ cells and oocytes [17]. Results from MTT assay showed that survival of ovarian cells was significantly decreased in higher PCBs-treatment (10 ␮g/ml). As decreases in MTT absorbance were interpreted as decreases in cell viability due to mitochondrial dysfunction [33], the MTT reduction might reflect the cell loss. PCB-induced decrease in MTT absorbance may also indicate inactivation of cytosolic

or microsomal enzymes involved in reduction of this indicator. The LDH leakage level was consistent with the results of MTT. In the groups with severe cell damage, the leakage of LDH was markedly higher than the control. These morphological and biochemical changes in ovarian cells manifested clear evidence of the direct toxicity of PCBs on reproduction. For estimation of the toxic effect of PCBs on ovarian cells, tocopherol was added for possible attenuation of the toxicity. The modality observation and assessments of ovarian cell damage both showed that the toxic effects of PCBs could be attenuated by tocopherol. The MTT reduction was higher in the combined treatment of PCBs with tocopherol than groups treated with PCBs alone. In all conditions the morphological estimate was consistent with the results obtained with the MTT assay. Ovarian cytotoxicity was also assayed by LDH release. The toxic effect of PCBs on LDH leakage was partly inhibited by tocopherol co-treatment. These results indicated that PCBs-mediated cell death might be induced by the formation of excessive free radicals, which led to lipid peroxidation and membrane impairment. For determination of estrogenic effect of PCBs, tamoxifen was used to block estrogen receptor activation in the process of PCBs action. In the initial hours after PCBs-treatment, toxic effect, but not significant estrogenic effect exhibited. In the latter period, PCBs elicited estrogenic action in promoting germ cell proliferation. The number of germ cell still increased in 10 ␮g/ml group despite the severe toxicity on ovarian cells in the former period. This result indicated that the estrogenic effect of PCBs appeared later after its toxic effect. Difference in time between these two effects was easy to interpret as the increment of germ cells by the estrogenic effect of PCBs took relatively longer time to finish cell cycles. The increment of germ cell number by PCBs was blocked by tamoxifen. Combined treatment of PCBs with tamoxifen showed only toxic effects on ovarian cells. The toxicity was even severer than PCBs-treatment alone. Many studies suggested that hydroxylation was important for interaction with estrogen receptor. Weak estrogenic activity of PCBs was observed in a number of studies [34]. However, it is conceivable that in some cases PCBs were metabolized in the test system to hydroxylated PCBs. The inhibiting effects of PCBs on germ cell development were further proved by the in vivo experiment in which PCBs inhibited germ cell proliferation and caused cell degeneration. The results by PCBs appeared to be dependent on the timing and dose of exposure, in which exposure during sex differentiation possibly altered subsequent fertility and secondary sexual characteristics [35]. Eggs injected after the period of organ development of the embryo resulted in substantially less embryo mortality and greater chick growth [36]. While if injected at the early incubation period, PCBs induced a dose-dependent decrease of primordial germ cell (PGC) numbers, and caused PGC pyknosis and plasma vacuolation via toxic rather than estrogenic effect [37]. And in the late phage of chicken embryo, Aroclor 1254 showed

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both toxic and estrogenic effects on ovarian developments [23]. In conclusion, the germ-somatic cell co-culture model offered a convenient method to investigate toxic and estrogenic effects of endocrine-disrupting chemicals on gonadal germ cells in vitro. PCBs caused dose- and time-dependent toxic and estrogenic effects on germ cell morphology and proliferation. In combination with these two discrete effects, PCBs imposed disrupting actions on ovarian germ cell development, including increased cell proliferation and decreased cell viability. The nuclear hyperchromatism induced by PCBs might result in genotoxicity of the germ cells, leading to infertility of the animals.

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