Toxicology Letters 183 (2008) 10–20

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Toxicology Letters

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NP-induced biophysical and biochemical alterations of rat testicular Sertoli cellmembranes related to disturbed intracellular Ca2+ homeostasis

Yi Gonga,b,1, XuPing Pana,b,1, Yufeng Huangc, ZiShen Gaod, HongXia Yud, Xiaodong Hana,b,∗

a Immunology and Reproductive Biology Laboratory, Medical School, Nanjing University, Nanjing 210093, PR Chinab Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing 210093, PR Chinac Department of Biochemistry, Jinling Hospital, Clinical School of Medicine, Nanjing University, Nanjing 210002, PR Chinad State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:Received 14 May 2008Received in revised form 29 July 2008Accepted 22 August 2008Available online 2 September 2008

Keywords:NonylphenolPharmacokineticsMembrane characteristicsFSHRATPaseCa2+

a b s t r a c t

Nonylphenol (NP) is a representative endocrine disruptor that has an adverse effect on male reproduc-tion and posses direct hazard to Sertoli cells, but the mechanism remains incompletely elucidated. Inthe present study, based on the structural comparability and high affinity between NP and membranephospholipid molecules, we tested the hypothesis that entrance of NP into Sertoli cells would alter mem-brane biophysical characteristics and biochemical functions. First, we used gas chromatography–massspectrometry (GC–MS) to investigate the distribution and pharmacokinetics of NP in Sertoli cells withthe result revealing that NP could penetrate plasma membrane of Sertoli cells. Meanwhile, Sertoli cellstreated with NP exhibited abnormal membrane potential; that is an early depolarization following shorttreatment and hyperpolarization after longer treatment with the highest concentration of NP. Studieson the membrane dynamics indicated that the NP exposure rendered increased membrane fluidity anddecreased microviscosity and molecular order. The result of lactate dehydrogenase (LDH) leakage assaydemonstrated that NP increased membrane permeability in time–dose-dependent manners. Atomic forcemicroscopic (AFM) imaging was applied to examine the membrane topography, and the images showedthat NP treatment caused disturbance of membrane topography. The activities of plasma membrane Ca2+-

2+ 2+ + +

ATPase, Ca –Mg -ATPase and Na –K -ATPase were also changed following NP exposure. However, FSHreceptor as an important membrane protein was not significantly altered. All the above changes led to

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. Introduction

NPE (nonylphenol polyethoxylate) is a nonionic surfactant ands widely used in the manufacture of detergents, herbicides, insec-icides, plastic additive and emulsifiers (White et al., 1994). Thennual output of NPE in the world is about 400,000 tons (Nalort al., 1996; de Voogt et al., 1997), most of which enters the waternvironment (Talmage, 1994). NPE has a low toxicity, but the com-

ound is not stable in the environment and can be biodegraded intoore stable but less water-soluble NP (Hawrelak et al., 1999). The

ipophilic feature of NP allows it to absorb to the surface of organ-sms and granules and then be discharged into rivers and sea by

∗ Corresponding author at: Immunology and Reproductive Biology Laboratory,edical School, Nanjing University, Nanjing 210093, PR China.

el.: +86 25 83686497; fax: +86 25 83686559.E-mail address: hanxd@nju.edu.cn (X. Han).

1 These authors contributed equally.

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378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2008.08.011

homeostasis which was an important signal triggering apoptosis. Hence,d a plausible target for NP-induced cytotoxicity.

© 2008 Elsevier Ireland Ltd. All rights reserved.

ewage (Ahel et al., 1994; Naylor et al., 1992; Giger et al., 1984). Itas reported that the concentration of NP in rivers of Chongqingity (China) had approached 1.7–7.2 mg/l (Shao et al., 2005). There-

ore, NP has become an important exposure source for animalsn aquatic ecosystems (Kannan et al., 2003). Due to its lipid sol-bility and stability, NP can also accumulate in the river sedimentnd in the fatty tissue of exposed aquatic organisms such as fish,hrimp and seashell fish (Shiraishi et al., 1989; Ekelund et al., 1990;hel et al., 1993). The bioaccumulation of NP in aquatic organisms

ncreases the latent exposure risk to higher vertebrates via the foodhain and may pose great harm to human and animals.

Over the past few decades, NP was demonstrated to exhibit tox-city and disrupting effect on the reproductive systems of malesh, amphibians and mammals (Nakamura et al., 2002; Weber et

l., 2002; Cardinali et al., 2004; Nagao et al., 2001). In our previ-us studies, we found that NP treatment resulted in great damageo the reproductive system, including altered steroidogenesis, dis-urbed testicular structure and decreased sperm number in thepididymis (Han et al., 2004). Our further study demonstrated that

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ertoli cells were one of the target cell types of NP. Exposure toP led to the morphological abnormality, decreased viability andven apoptosis and necrosis of Sertoli cells (Gong and Han, 2006;ang et al., 2003). As the only somatic cells in the testicular sem-

niferous epidermis, Sertoli cells create a special morphologicalnd biochemical environment that is essential for the attachmentnd development of germ cells; they also provide a great dealf nutrients and hormones needed by the germ cells (Griswold,998). Therefore, alteration of Sertoli cells will lead to impairedpermatogenesis, germ cell loss and male infertility (Monsees etl., 2000). Hence, study on the mechanism of NP-action on Ser-oli cells is important for investigating NP-induced reproductiveoxicity.

As we all know, Ca2+ is an essential intracellular second mes-enger and its concentration is maintained in normal cells. Hence,isturbance of Ca2+ homeostasis may trigger diverse cellular pro-esses, even apoptosis (Annunziato et al., 2003). Earlier studiesndicated that NP treatment caused intracellular Ca2+ elevation ineveral cell types, including human osteosarcoma cells and TM4ells (Sertoli cell line) (Hughes et al., 2000; Wang et al., 2005),hough the detailed mechanisms of NP-induced intracellular Ca2+

levation are still unknown.Plasma membrane constitutes the boundary between a living

ell and its environment, and plays an important functions inontrolling the transport of ions. Disturbance of the membranetructure as well as changes of membrane characteristics will leado functional alteration of the membrane, particularly the trans-ort and flux of Ca2+ through membrane channels, which mayrigger the apoptotic signal pathway. The molecular structure ofP consisting of a hydrophilic OH and a lipoophilic alkyl-benzeneomain resembles the structure of membrane phospholipid, andhis characteristic of NP determined its high affinity for membranehospholipid. It was reported by Meier et al. (2007) that NP hadn especially great effect on both surface tension and the meanolecular area of the phospholipid monolayer. We speculate that

uring the entrance process into Sertoli cells, NP can interact withhe membrane phospholipids or proteins, rendering the alterationf biological and physical characteristics of the plasma membranend subsequently leading to the disturbance of cellular homeostasisnd even apoptosis.

In the present study, we used gas chromatography–masspectrometry (GC–MS) to first investigate the distribution andharmacokinetics of NP in Sertoli cells. Then, several impor-ant membrane biophysical characteristics were examined afterP treatment. In order to further explore the NP-action on bio-hemical functions of the membrane, we examined the changesf Ca2+-ATPase, Ca2+–Mg2+-ATPase and Na+–K+-ATPase activi-ies and the important membrane receptor, follicle-stimulatingormone receptor (FSHR). In addition, intracellular free Ca2+

lteration in Sertoli cells following NP exposure was also exam-ned.

. Material and methods

.1. Chemicals and reagents

NP (4-nonylphenol) with 98% analytical standard and butylphenol wereurchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Bis(trimethylsilyl)rifluoroacetamide (BSTFA) used as derivatizing reagent was from Supelco (Belle-onte, PA, USA). The hexane of pesticide grade was obtained from Tedia Companync. (Fairfield, OH, USA). Dulbecco’s modified Eagle’s medium-Ham’s F-12 mediumDMEM-F12 medium), penicillin, streptomycin sulfate, trypsin, collagenase I, 1,6-

iphenyl-1,3,5-hexatriene (DPH) and bovine serum albumin (BSA) were purchasedrom Sigma–Aldrich Inc. (St. Louis, MO, USA). C8H17N2O4SNa (HEPES sodium salt)as obtained from Amresco Inc. (Solon, OH, USA). Bis-1,3-dibutylbarbituric acid-

rimethine oxonol (DiBAC4(3)) was bought from Invitrogen Inc. (Eugene, OR,SA). Fluo 3 AM was obtained from AnaSpec Inc. (San Jose, CA, USA). Anti-SHR antibody and donkey anti-goat IgG-FITC were purchased from Santa Cruz

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ters 183 (2008) 10–20 11

iotechnology Inc. (Santa Cruz, CA, USA). Lactate dehydrogenase (LDH) detec-ion kit, in addition with Ca2+-ATPase, Ca2+–Mg2+-ATPase and Na+–K+-ATPasessay kits were from Jiang Cheng Bioengineering Institute (Nanjing, Jiangsu,hina).

The culture medium used was DMEM-F12 (1:1) supplemented with 4 mM glu-amine, 15 mM hepes, 6 mM l-(1)-lactic hemicalcium salt hydrate, 1 mM sodiumyruvate, antibiotics (final concentrations: penicillin, 100 IU/ml; streptomycin sul-ate, 100 �g/ml).

.2. Isolation and culture of Sertoli cells

Sprague-Dawley Rats were purchased from Nanjing Medical University and keptn accordance with NIH Guide for the Care and Use of Laboratory Animals. Ser-oli cells were isolated from rats at the age of 30 days according to the method ofteinberger et al. (1975) with some modifications. Testes were removed and decap-ulated. Six testes were placed in a 50-ml conical tube in 40 ml PBS, washed twice,nd allowed to settle. The seminiferous tubules were dispersed gently using forceps,ut not fragmented, and then washed twice again in PBS. The settled seminiferousubules were incubated with 0.25% trypsin at 37 ◦C, shaking at 50 oscillation/minor about 30 min. Care was taken not to fragment the seminiferous tubules duringrypsin digestion, because tubule fragmentation results in poor yield and purity.he supernatant, which contained interstitial cells, was decanted. The tubules wereashed three times (40 ml PBS) and then incubated in a solution (20 ml) con-

aining 0.1% collagenase at 37 ◦C, shaking at 80 oscillations/min for 40 min. Thereparation then was filtrated through a 100-mesh stainless steel filter, and col-

ected for centrifugation at 1000 rpm for 6 min. The pellet was subsequently washedhree times (40 ml PBS). At this step of the isolation procedure, the Sertoli cellsere single cells, and with germ cells as the major contaminating cell type. Cellsere resuspended in DMEM-F12 medium containing 5% fetal bovine serum (FBS)

nd seeded onto culture flasks and maintained in a humidified atmosphere of5% air, 5% CO2 at 34 ◦C. In the next steps, we separated Sertoli cells and germells by their different ability of attachment. After 2 days in culture, Sertoli cellsttached to the bottom of flasks with only tiny dendrites protruding, but most oferm cells suspended in the medium were removed by changing the medium. Theemaining germ cells were hypotonically lysed with 20 mM Tris–HCl (pH 7.4) treat-ent. Two days later, the medium was changed again for the second purification

nd the Sertoli cells with high purity grew quickly to form a monolayer in newedium.

.3. Sertoli cells treatment with NP

NP was dissolved in DMSO and diluted to different concentrations using DMEM-12, with the concentration of DMSO less than 1/1000 and consistent in each group.ultured Sertoli cells were treated with different concentrations of NP for indi-ated time periods, and then several parameters about membrane alterations wereetected as follows.

.4. Cell fraction preparation and extraction of NP

During the experiment, Sertoli cells treated with NP for designated time periodsere collected by trypsinization and centrifugation. Afterwards, aliquots of cells inifferent groups were adjusted to the same density (1.5 × 106/ml) and homogenizedy ultrasonic treatment. All the prepared samples were centrifuged in 10,000 rpm for0 min in order to remove cell fragments. Then the pH of the solution was adjustedo 2–3 by adding HCl (0.5 M). Liquid–liquid extraction was adopted to extract theP in the solution by adding about 20 ml hexane and shaking for several minutes.he hexane was separated by centrifugation and the extraction was repeated threeimes to ensure that all the NP was extracted. All the hexane was collected andvaporated to almost dry by a rotary evaporator. The concentrated hexane was thenransferred to a glass vial. After spiking 500 ng internal standard, 4-n-butylphenol,ll the liquid was concentrated to near dryness under gentle nitrogen gas. After this,.1 ml BSTFA was added into the tube and the derivatization was incubated at 65 ◦Cater bath for 30 min. After the reaction, the volume of the reagent was adjusted

o a final volume of 500 �l by adding hexane and the sample was stored at −20 ◦Crior to analysis.

.5. Gas chromatography–mass spectrometry (GC–MS)

A Finnigan GCQ interfaced with an ion trap MS detector (Finnegan, Amer-ca) equipped with a HP DB-5 column (30 m × 0.25 mm× 0.25 �m) (Fluka, Buchs,witzerland) was used for the sample analysis. Manual injection of 1 �l sam-les was conducted at the splitless mode. The analysis was under scan modend the scan ion m/z was from 50 to 350. The inlet temperature of the GC was

80 ◦C. The temperature of the transfer line and ion source temperature wereet to 260 and 210 ◦C, respectively. The carrier gas was helium at 40 cm/s. Theven temperature program was as follow: initial temperature was 80 ◦C, thenncreased to 220 ◦C at 12 ◦C/min and held for 2 min, then increased to 300 ◦C at5 ◦C/min and held for 12 min. The identification of NP is based on the ion frag-ents in the mass spectrum. The quantitative analysis was performed by monitoring

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on fragments m/z 197, m/z 292 and m/z 197, m/z 222 for NP and butylphenol,espectively. Quantification of NP was carried out with the internal calibrationrocedure.

.6. Measurement of plasma membrane potential

The membrane-potential-sensitive dye DiBAC4(3) is widely used in determin-ng the changes in membrane potential. The distribution of this probe within the

embrane and the resulting fluorescence is dependent on the transmembrane

otential of the cell (Tanner and Wellhausen, 1998). A linear relationship betweenembrane potential and the DiBAC4(3) fluorescence in various cell types has been

stablished (Epps et al., 1994). In the present study, Sertoli cells were incubatedn the presence of different concentrations of NP for various durations, cells then

ere loaded with 4 �M DiBAC4(3) for 20 min at 37 ◦C and the related fluorescenceas observed under the fluorescent microscopy (Nikon, Chiyoda-ku, Tokyo, Japan)

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ig. 1. Distribution and pharmacokinetics of NP in Sertoli cells. After exposure to 10 anomogenized by ultrasonic. Concentrations of NP in cell fractions was examined by GC–Mf NP standard (m/z = 179,222 and m/z = 179,292). (B) Total ion current chromatograph anutylphenol ramification (upper) and NP ramification (lower). (D) Concentrations of NP in

ters 183 (2008) 10–20

nd recorded using a multi-detection microplate reader (Bio-TEK, Highland Park,T, USA) at 488 nm.

.7. Plasma membrane dynamics determination

Membrane dynamics was determined continuously by measuring fluorescencenisotropy in intact cells by using DPH as a probe (Chu-Ky et al., 2005; Konopásekt al., 2004). The determination was optimized as follows: Sertoli cells treatedith increasing concentrations of NP were collected and then incubated with

�M DPH for 30 min at 37 ◦C. Steady state fluorescence polarization measure-ents were performed with a Hitachi 850 spectrofluorimeter using a Hitachi

olarization accessory with Quartz cuvettes of 1 cm path length. The excitationavelength was set at 357 nm and emission was monitored at 430 nm. Excitation

nd emission slits with bandpass of 10 and 7 nm were used for all measure-ents.

d 20 �M NP for 3, 6, 12 and 24 h, Sertoli cells in each group were collected andS. (A) Total ion current chromatograph and selected ion monitoring chromatogramd selected ion monitoring chromatogram of NP in samples. (C) Mass spectrum ofSertoli cell fractions.

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maiuNP groups (0.1 and 1 �M). Fig. 5 represents alteration of mem-brane dynamics of Sertoli cells following exposure to 30 �M fordifferent times. With the increased exposure time, an augmentedmembrane fluidity, decreased microviscosity and molecular orderwere detected, and the effect was more pronounced at 12 and 24 h.

Y. Gong et al. / Toxicolo

Polarization values and membrane dynamics parameters were calculated fromhe equations (Chu-Ky et al., 2005; Arora et al., 2004; Murata et al., 2007).

= IVV − GIVH

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olecular order : r = 2P

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here IVV and IVH are the measured fluorescence intensities with the excitationolarizer oriented vertically and the emission polarizer vertically and horizontallyriented, respectively. G is the grating correction factor and is the ratio of the effi-iencies of the detection system for vertically and horizontally polarized light, ands equal to IHV/IHH. All experiments were done with multiple sets of samples.

.8. Plasma membrane permeability detection

Extracellular LDH levels were used as an indicator of membrane permeabilityOlabarrieta et al., 2001). Briefly, Sertoli cells were seeded in 96-well plates andxposed to various doses of NP for indicated times. LDH leakage in culture mediumas measured using LDH detection kit according to the manufacturer’s instructions.

.9. Atomic force microscope (AFM) imaging

Sertoli cells were seeded into six-well plates in which clean cover slips hadeen placed. After treatment with 20 and 30 �M NP for indicated times, cover slipsere gently rinsed with PBS to remove loosely attached cells and then fixed in 2%

lutaraldehyde for 5 min. Afterwards, cover slips were washed twice with PBS andir-dried. Atomic force microscopy (AFM) imaging was performed on a MultimodeFM (Agilent 5400, Agilent Inc., USA) operated in a tapping mode. Measurementsere carried out using silicon cantilevers with a spring constant of 20 N/m and a res-

nance frequency of 150 kHz. Images were acquired at a scan field of 10 �m × 10 �mr 30 �m × 30 �m.

.10. Immunofluorescence analysis by flow cytometry

Sertoli cells exposed to various concentrations of NP for 24 h were removed fromlates by treating with 0.02% EDTA in PBS. Cells were collected by centrifugation andashed twice in PBS, and resuspended in PBS containing 1% FBS at 37 ◦C for 30 min,

hen harvested by centrifugation. The cells were incubated at 37 ◦C for 2 h with therimary anti-FSHR antibody. To block nonspecific binding, the primary antibody wasiluted 1:100 with 1% BSA in PBS. Following incubation with the primary antibody,he cells were then incubated at 37 ◦C for 1 h with FITC-conjugated secondary anti-ody diluted in 1% BSA–PBS. The cells were then washed and resuspended in PBS.ell surface immunofluorescence was measured using a FACScan flow cytometerBecton–Dickson, San Jose, CA, USA).

.11. ATPase activity assay

Sertoli cells treated with 10 and 20 �M NP for different times were harvestednd resuspended in ST buffer (10 mM Tris–HCl, 250 mM sucrose, pH 7.5), then frag-entated by ultrasonic, and diluted 10 times with ST buffer. The activities of plasmaembrane Ca2+-ATPase, Ca2+–Mg2+-ATPase and Na+–K+-ATPase were estimated by

uantifying the release of inorganic phosphorus from adenosine triphosphate (ATP)ccording to the manufacturer’s instructions of assay kits. One activity unit of ATPaseas expressed as 1 �mol phosphate per milligram protein per hour. All of the

olorimetry assays were measured on an automated microplate reader (Bio-TEK,ighland Park, VT, USA) at 636 nm.

.12. Intracellular free Ca2+ detection

Intracellular Ca2+ detection of Sertoli cells was performed after cells werexposed to NP for certain time. In brief, cells planted into 96-well plates were loadedith 4 �M Fluo-3 AM in PSS buffer (136 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1 mMgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 37 ◦C for 50 min. The resulting

uorescence as the indicator of Ca2+ concentration was observed under the fluores-ent microscopy (Nikon, Chiyoda-ku, Tokyo, Japan) at 488 nm excitation wavelength

nd analyzed by using the software of SimplePCI.

.13. Statistical analysis

All the experiments were replicated at least three times. Results were showns means ± S.D. Statistical evaluation of the data was performed by using one-way

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ters 183 (2008) 10–20 13

nalysis of variance (ANOVA) technique in SPSS followed by Tukey test. P < 0.05 wasccepted as the minimum level of significance.

. Results

.1. Distribution and pharmacokinetics of NP in Sertoli cells

In order to investigate the distribution and pharmacokineticehavior of NP in Sertoli cells, primary cultured Sertoli cells werereated with 10 and 20 �M NP for 3, 6, 12 and 24 h, respectively, andomogenized by ultrasonic treatment. NP in the cell fractions wasxtracted by the procedure described in Section 2, and then exam-ned using GC–MS. NP concentration in Sertoli cells of both 10 and0 �M groups gradually increased with prolonged exposure time to2 h. However, after 24 h exposure, NP in both groups dramaticallyecreased to the level at 3 h (Fig. 1.).

.2. Membrane potential alteration

As shown in Fig. 2, addition of graded concentrations of NP toertoli cells for 1 and 3 h caused significant increase of DiBAC4(3)uorescence from even the lowest concentration of NP, indicat-

ng depolarization of the membrane. Fig. 3 shows the fluorescentmages of Sertoli cells loaded with DiBAC4(3) following NP expo-ure for 3 h, which indicate increased fluorescence in NP-treatedroups as compared with control. With the exposure time extendedo 6, 12 and 24 h, the DiBAC4(3) fluorescence in 0.1, 1, 10 and 20 �MP groups came back to normal level, though still higher than theontrol, implying the recovery of the membrane potential. How-ver, at the highest concentration (30 �M), DiBAC4(3) fluorescenceeclined dramatically from 6 to 24 h, suggesting the irreversibleyperpolarization of the membrane.

.3. Changes of membrane dynamics

Fig. 4 shows the effect of increasing concentrations of NP onembrane dynamics involving membrane fluidity, microviscosity

nd molecular order of Sertoli cells. 10, 20 and 30 �M NP caused anncreased membrane fluidity, decreased microviscosity and molec-lar order. However, there was no significant effect in the lower

ig. 2. NP-induced membrane potential alteration. Sertoli cells exposed to differentmounts of NP (0, 0.1, 1, 10, 20 and 30 �M) for 1, 3, 6, 12 and 24 h were loaded withiBAC4(3) and the relative fluorescence which represents the membrane potentialas measured. Data are presented as means ± S.D. Statistically different from the

ontrol is marked with asterisk (*P < 0.05 and **P < 0.01).

14 Y. Gong et al. / Toxicology Letters 183 (2008) 10–20

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ig. 3. Fluorescent images of Sertoli cells loaded with DiBAC4(3). Sertoli cells treaere loaded with DiBAC4(3) and photographed under fluorescent microscope. Bar =

.4. LDH assay for membrane permeability detection

Release of LDH into the culture medium was measured to assessP effect on the membrane permeability of Sertoli cells. Fig. 6 shows

hat NP increased membrane permeability in both time and dose-ependent manners. After 6 h treatment, only 30 �M NP increasedembrane permeability. When the exposure time prolonged to 12

nd 24 h, 10, 20 and 30 �M NP all resulted in significant increase ofembrane permeability with higher concentrations inducing more

ignificant effect.

.5. AFM imaging for determining membrane topography

In order to gain insight into the fine structure of Sertoli cellsreated with NP, the local structure of the cell surface was examinedy zooming at the regions of 30 �m × 30 �m or 10 �m × 10 �m.

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th 0 �M (A), 0.1 �M (B), 1 �M (C), 10 �M (D), 20 �M (E) and 30 �M (F) NP for 3 hm.

he AFM images of Sertoli cells revealed a remarkable differ-nce in cell surface architecture after treatment with 20 and0 �M NP (Fig. 7). The white areas in the images indicate therotuberance in the surface of the membrane while the darkreas indicate the concaves. From the 30 �m × 30 �m pictures,t was observed that the membrane surface of Sertoli cells inhe control was smooth with protuberance and concaves evenlyistributed (Fig. 7a–d), whereas the membrane of NP-treated Ser-oli cells was occupied with many concentrated protuberanceFig. 7b, c, e and f). In the 10 �m × 10 �m images, Sertoli cellsreated with NP exhibited higher degrees of surface roughness

ith rounded, oval, triangular or quadrangular perikarya fromhich different degrees of thick dendrites arose (Fig. 7h and i),hile untreated cells were featured by smooth contours with few

mall ‘crater-like’ pits and indentations spread over cell surfaceFig. 7g).

Y. Gong et al. / Toxicology Letters 183 (2008) 10–20 15

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ig. 4. NP-induced changes of membrane dynamics. After treatment with different concrder (C) of Sertoli cells were measured as described under Section 2. Data are present*P < 0.05 and **P < 0.01).

ig. 5. Time-dependent effect of NP on membrane dynamics. Following exposure to 30 �nd molecular order (C) were measured. Data are presented as means ± S.D. Statistically d

entrations of NP for 24 h, membrane fluidity (A), microviscosity (B) and moleculared as means ± S.D. Statistically different from the control is marked with asterisk

M NP for 0, 6, 12 and 24 h, alteration of membrane fluidity (A) microviscosity (B)ifferent from the control is marked with asterisk (*P < 0.05 and **P < 0.01).

16 Y. Gong et al. / Toxicology Let

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ig. 6. LDH assay for membrane permeability detection. After Sertoli cells werereated with 0, 0.1, 1, 10, 20 and 30 �M for 6, 12 and 24 h, LDH in the culture mediumas detected. Data are presented as means ± S.D. Statistically different from the

ontrol is marked with asterisk (*P < 0.05 and **P < 0.01).

.6. Immunofluorescence analysis of FSHR

Immunofluorescence combined with flow cytometry was usedo evaluate the protein level of FSH receptor (FSHR) on the plasma

embrane. Sertoli cells treated with graded concentrations of NPor 24 h were harvested for the detection. A slight decrease of FSHRevel was observed in NP-treated groups, but there was no signifi-ant difference in comparison to the control (Fig. 8.).

.7. ATPase analysis

The effect of NP on the activity of Ca2+-ATPase, Ca2+–Mg2+-TPase and Na+–K+-ATPase was evaluated after Sertoli cells werereated with 10 and 20 �M NP for 0, 3, 6, 12 and 24 h, respec-ively. The activity of Ca2+-ATPase was significantly increased after

and 6 h treatment with both 10 and 20 �M NP, but dramat-cally decreased after 12 and 24 h exposure (Fig. 9A). Changesf Ca2+–Mg2+-ATPase showed a similar tendency, though notignificant at 12 h versus the control (Fig. 9B). The activity ofa+–K+-ATPase increased dramatically to the maximum at 3 h

ollowing 10 �M NP exposure, and declined gradually with thelongation of time, though not significantly. However, in 20 �M NProup, the activity of Na+–K+-ATPase exhibited a slight elevationt 3 and 6 h, and then significantly decreased both at 12 and 24 hersus control (Fig. 9C).

.8. Intracellular free Ca2+ studies

The result shown in Fig. 10 illustrated that NP induced a dose-ependent elevation of cytosolic free Ca2+. The differences betweenhe NP-treated groups and the control were all significant after 2 hxposure. When exposure time increased to 6 h, Ca2+ concentra-ions in NP-treated cells were still higher than that in the control,ut only 10, 20 and 30 �M treatments exhibited significant differ-nces as compared with the control.

. Discussion

Spermatogenesis is an enormously complex and dynamic pro-ess during which numerous cellular interactions involving Sertoliells, germ cells, and the Leydig cells allow for attachment androwth of the germ cells into haploid sperm. The Sertoli cell thatccupies a significant portion of the long tubular space is highly

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ters 183 (2008) 10–20

rregular in shape, extending from the basal lamina throughouthe way into the tubular lumen. This unusual structure is thoughto provide the scaffolding necessary for the anchoring and nour-shment of the germ cell and any impairment to the structure

ill pose great hazard to the entire spermatogenesis (Griswold,998; Sairam and Hanumanthappa, 2001). Our current studyemonstrated that NP could enter Sertoli cells and induced signifi-ant alterations of membrane biophysical characteristics, includingembrane potential, dynamics, as well as membrane permeability

nd membrane topography. In addition, NP treatment also resultedn activity changes of several important membrane proteins suchs Ca2+-ATPase, Ca2+–Mg2+-ATPase and Na+–K+-ATPase. However,he expression of Sertoli cell-specific FSHR, which is also situated atlasma membrane and exerts important functions in cell signalingystem, was only slightly influenced.

Though NP has been extensively studied on account of itside-range existence in the environment, little is known about

ts distribution and pharmacokinetic behavior in mammalian cells,specially in gonad cells. So far, it was only reported by Müller et al.1998) that NP could be rapidly taken up by primary mouse hepa-ocyte cells. In current study, we demonstrated that NP could enterertoli cells with its concentration in cell fractions showing an ini-ial increase and a final decline. We considered several possibleeasons for the occurrence of the final decline: (a) NP was partlyetabolized by Sertoli cells. For example, Müller et al. demon-

trated that NP could rapidly be metabolized in hepatocyte cells,hich led to the decrease of NP in cell fractions. (b) NP was con-

ugated with other cellular components, leading to the dramaticecrease of the parental NP. This is in agreement with the stud-

es of Certa et al. (1996) showing that microsomal enzymes areapable of conjugating AP in vitro. (c) NP was lost during the prepa-ation of cell samples due to increased membrane permeability.s was addressed in the results, membrane permeability was sig-ificantly increased by 24 h NP-treatment, and this may help theasier leakage of NP during the subsequent preparation of cellamples.

As noted above, the entrance of NP into Sertoli cells is affirmativend it is clear that the first encounter with NP on the cellular levels the plasma membrane. Meier et al. (2007) has illustrated thatP treatment caused an alteration of membrane tension and an

ncrease in the mean molecular area per number of phospholipidn the membrane. We speculate that during the process of entrynto cells, amphiphilic NP can penetrate the lipid–water boundaryf the membrane by inserting the lipophilic alkyl-benzene domainmong the fatty acid moiety of glycerophospholipids in the mem-rane and the hydrophilic OH groups being positioned among theydrophilic phospholipid headgroups, finally rendering alterationf membrane characteristics.

To investigate the mechanisms, we first measured the mem-rane potential changes. As was expected, alteration of plasmaembrane potential was observed after NP treatment, and thatas an early depolarization in all NP groups and hyperpolariza-

ion after longer time treatment of the highest NP. Previous datallustrated that many organochlorine insecticides such as DTT andelated compounds extended the Na+ current in axon membranes,hile Lindane and compounds of cyclodiene family affected thea2+ permeability of presynaptic membranes and the influx of Cl−

n postsynaptic membranes (Clark, 1997; Doherty, 1979). In ourtudy, elevated intracellular Ca2+ concentration was also observed,hich demonstrated that ion fluxes were altered, and this will def-

nitely cause the disruption of membrane potential. Meanwhile,he change of membrane potential will also induce the openingf voltage-gated ion channels in the plasma membrane and finallyead to subsequent ion flux which may disturb the cellular home-stasis.

Y. Gong et al. / Toxicology Letters 183 (2008) 10–20 17

F cells tru ted wi3

pfl1t(dflabwblioM

mi

baaItlh

ig. 7. Atomic force microscope images showing the membrane topography. Sertolising atomic force microscope under 30 �m × 30 �m fields of view. Sertoli cells trea0 �m × 30 �m (upper) and 10 �m × 10 �m (below) fields of view.

Optimal membrane dynamics is essential in maintaining normalhysiological functions. Many investigations show that membraneuidity can be affected by xenobiotics like lindane (Suwalsky et al.,998), �- and �-endosulfan (Videira et al., 1999), dichlorodiphenyl-richloroethane (DDT) (Nelson, 1987), polyaromatic hydrocarbonsPAHs) (Engelke et al., 1996; Jimenez et al., 2002) and 2,4-ichlorophenol (Csiszar et al., 2002). In the present study, DPH asuorescent probes was used for studying the membrane dynamics,nd changes of membrane dynamics including increased mem-rane fluidity and decreased microviscosity and molecular orderere also observed following NP exposure. These changes might

e partly attributed to the direct action of NP. Besides, we specu-

ated that the disruption of plasma membrane potential and alteredntracellular ion levels such as Ca2+ can cause changes in activityf membrane proteins and lead to membrane dynamic changes.eanwhile, change of membrane dynamics in a cell will lead to dra-

bco2S

eated with 20 �M NP for 0 h (a), 12 h (b) and 24 h (c) were processed and examinedth 30 �M NP for 0 h (d and g), 12 h (e and h) and 24 h (f and i) were examined under

atic alterations in ion regulation and normal cellular physiologyn a feedback way.

Previous reports have demonstrated that alterations of mem-rane fluidity can lead to altered membrane permeability (Drori etl., 1995; Bengoechea et al., 2003). Results from the current studylso showed augmented membrane permeability induced by NP.ncreased membrane permeability can produce a breakdown ofransmembrane ion gradients and inhibition of integrated cellu-ar metabolic processes (Muriel and Sandoval, 2000), which mightelp explain the hyperpolarization after prolonged NP treatment.

Recently, AFM is emerging as a powerful tool for investigating

iological processes and topographic structures on the surface ofells and biopolymers, providing three dimensional molecular res-lution of biological molecules (Hoh and Hansma, 1992; Dufrêne,002). To further characterize membrane injury induced by NP,ertoli cells were planted on cover slips and imaged by AFM in

18 Y. Gong et al. / Toxicology Letters 183 (2008) 10–20

F ted wd muno

towttf

Cb

edio

Fh(

ig. 8. Immunofluorescent analysis of FSHR after NP exposure. Sertoli cells were treaetection on flow cytometry. (A) Flow cytometry plot of different NP groups. (B) Im

he tapping mode. Compared with the relative smooth contoursf untreated Sertoli cells, protruding particles with different shapesere found to occupy most of the membrane surface in Sertoli cells

reated with NP, indicating a disorganized topographic structure of

he membrane. These AFM images provided an additional evidenceor the toxic effects of NP on Sertoli cells membrane.

In addition, three important membrane enzymes, Ca2+-ATPase,a2+–Mg2+-ATPase and Na+–K+-ATPase were markedly influencedy NP treatment and exhibited a similar alteration trend, i.e., first

libNp

ig. 9. NP-induced alterations of Ca2+-ATPase, Ca2+–Mg2+-ATPase and Na+–K+-ATPase acarvested for Ca2+-ATPase (A), Ca2+–Mg2+-ATPase (B) and Na+–K+-ATPase (C) detection. Da* and #P < 0.05; **and ##P < 0.01).

ith control, 0.1, 1, 10, 20 �M NP for 24 h and then collected for immunofluorescencefluorescent analysis result of FSHR. Data are presented as means ± S.D.

levation after short-term treatment (3 and 6 h) and subsequentramatic decrease after longer time treatment (12 and 24 h). The

nitial elevation might have a close relationship with the changef membrane potential. As discussed above, short-term treatment

ed to the depolarization of the membrane, indicating a relativencrease of positive charges on the intracellular side of the mem-rane. Hence, the activities of Ca2+-ATPase, Ca2+–Mg2+-ATPase anda+–K+-ATPase might be elevated to help the outward transport ofositive ions. However, the final decrease in the activity of these

tivity. Sertoli cells treated with 0, 10 and 20 �M NP for 0, 3, 6, 12 and 24 h wereta are presented as means ± S.D. Statistically different from the control was marked

Y. Gong et al. / Toxicology Let

Fig. 10. Intracellular free Ca2+ alteration following NP exposure. Sertoli cells planteditlm

Ao

ifCpcCiitb1ii

taacwtmb

C

A

eY

R

A

A

A

A

B

C

C

C

C

C

d

DD

D

E

E

E

G

G

G

H

H

H

H

n 96-well plates were treated with 0, 0.1, 1, 10, 20 and 30 �M NP for 2 and 6 h, andhen loaded with Fluo-3 AM. The resulting fluorescence of Fluo-3 indicated the Ca2+

evel. Data are presented as means ± S.D. Statistically different from the control isarked with asterisk (*P < 0.05 and **P < 0.01).

TPases might be attributed to the disturbance of the membranerganization.

In our present study, NP yielded a considerable increase inntracellular Ca2+ levels, and the result was consistent with thoserom Wang study (Wang et al., 2005). The rise in intracellulara2+ level was highly associated with the change of membraneotential which could induce the opening of voltage-gated Ca2+

hannels in the plasma membrane and the subsequent influx ofa2+. Besides, modification of membrane dynamics and permeabil-

ty as well as the changes of ATPases might also cause the disruptionn intracellular Ca2+ homeostasis. It has been largely consideredhat abnormal rise of cytosol Ca2+ is directly or indirectly responsi-le for a series of abnormalities within cells (Orrenius and Nicotera,994; Jurma et al., 1997; Tagliarino et al., 2001). Therefore, NP-nduced elevation of intracellular Ca2+ levels might play key rolesn the final cytotoxicity of Sertoli cells.

In conclusion, we demonstrated that entrance of NP into Ser-oli cells could cause changes of membrane characteristics as wells functions; all these changes were closely interrelated and canffect each other in turn, which may finally result in the disturbed

ellular homeostasis and perhaps trigger the apoptotic signal path-ay. This study indicates possible directions for exploration into

he toxic mechanism of various environmental toxicants. However,ore direct studies are further required to validate the interactions

etween NP and the plasma membrane.

J

ters 183 (2008) 10–20 19

onflicts of interest

None declared

cknowledgements

The work was supported by the program of National Natural Sci-nce Foundation of China (20577019). The authors wish to thankajing Wang and Dongmei Li for their help in the experiment.

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