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Reproductive Toxicology 30 (2010) 377–386

Contents lists available at ScienceDirect

Reproductive Toxicology

journa l homepage: www.e lsev ier .com/ locate / reprotox

olecular mechanisms of isocyanate induced oncogenic transformation invarian epithelial cells

orantla Venkata Raghurama,b, Neelam Pathaka, Deepika Jaina, Hariom Panwara,emant Pandeya, Subodh Kumar Jainc, Pradyumna Kumar Mishraa,b,∗

Bhopal Memorial Hospital & Research Centre, Bhopal, IndiaDivision of Translational Research, ACTREC, Tata Memorial Centre, Navi Mumbai, IndiaDepartment of Biotechnology, Dr. HS Gour University, Sagar, India

r t i c l e i n f o

rticle history:eceived 4 February 2010eceived in revised form 14 May 2010ccepted 28 May 2010vailable online 8 June 2010

a b s t r a c t

Ovarian surface epithelium is under constant physiological pressure to maintain its integrity. Environ-mental toxic exposure can contribute to degenerative pathologies including ovarian cancer. Based on ourcurrent understanding, we aimed at listing mechanistic insights that contribute to ovarian carcinogenesisafter exposure to methyl isocyanate, an ubiquitous environmental pollutant. Ovarian epithelial cells man-ifested a persistent DNA damage response along with increased accumulation of GADD45, p21, p16INK4A

eywords:eproductive Toxicityarcinogenesisethyl isocyanate

enescence

and pRb proteins upon treatment. Increase in cell size and �-gal positive staining showing inception ofpremature senescence with morphological transformation and structural and numerical chromosomalabnormalities were also observed. Immuno-FISH analysis illustrated early loss of TRF2 protein suggestiveof telomeric dysfunction due to premature senescence and plausible association with chromosomal andmicrosatellite instability. Soft-agar assay displayed neoplasticity in treated cells demonstrating onset of

. Thead to

NA damage repairenomic instability

malignant transformationproliferation and might le

. Introduction

Ovary is a complex organ with widely differing cellular com-onents, including germ cells, granulosa cells, stromal cells, andhe ovarian surface epithelium (OSE). Toxicants impact each ofhese components differently through diverse cellular pathways.he mammalian ovary is covered by a singular layer of epithe-ial cells. The rather unassuming character of the ovarian surfacepithelium has generally inclined scientific community to ques-ion its functional significance. It has been stated that every ovarianycle involves disruption and regeneration of the ovarian epithe-ium, which is potentially mutagenic. Exposure to environmentaloxicants such as organic dusts, aromatic amines, aliphatic andromatic hydrocarbons have been shown to cause abnormal pro-

iferative effect on OSE and suggested to be etiologic agents forvarian cancer [1,2]. On the other hand, environmental toxicantsave also been attributed to accelerate the transition to reproduc-ive senescence which could be pathological and undesirable [3].

∗ Corresponding author. Present address: Division of Translational Research,dvanced Centre for Treatment, Research & Education in Cancer (ACTREC), Tataemorial Centre, Kharghar, Navi Mumbai 410 210, India.

el.: +91 022 27405121/27740520; fax: +91 22 27405061.E-mail address: pkm 8bh@yahoo.co.uk (P.K. Mishra).

890-6238/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2010.05.087

se results indicate that isocyanate exposure alters ovarian epithelial cellovarian dysfunction and carcinogenesis.

© 2010 Elsevier Inc. All rights reserved.

Stress-induced senescence is generally premature in nature.Recent findings have demonstrated that a phenotype called ‘stress-induced premature’ senescence (SIPS), can be induced withoutextensive cell division in normal as well as cancer cells by a vari-ety of stresses and signaling imbalances [4]. Cells undergoing SIPSmanifest the major characteristics that include an enlarged flat-tened morphology and positive staining for senescence-associated(SA) �-galactosidase activity. Increasing evidence also suggests thatthe process of replicative senescence can be prematurely inducedthrough a variety of stress-inducing agents, e.g. hyperoxia or UVirradiation. Rapid shortening of telomeres is also associated withSIPS [5]. The findings, that senescent cells acquire multiple pheno-typic changes and thereby compromising the tissue structure andfunction, have led to the hypothesis of senescence response as anexample of evolutionary antagonistic pleiotropy [6].

Evidence accumulated from previous studies has shown thatDNA damage check points are permanently activated in cells underexogenic influence with initiation of sensitive double strandedbreaks (DSBs) serving as a signal for SIPS. Incidentally, growtharrest DNA damage (GADD) 45 protein has been implicated in

stress signaling responses to various physiological or environmen-tal stressors, mediating their activity through a complex interplayof cellular proteins that are implicated in cell cycle such as pro-liferating cell nuclear antigen (PCNA), p21, cdc2/cyclinB1, andthe p38 and JNK stress response kinases [7,8]. However, the

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78 G.V. Raghuram et al. / Reprodu

olecular nature of stress response pathways involving GADD45nd its interacting proteins needs a further perusal.

The induction of intracellular proliferative signal in the arrestedells in combination with the blockade of apoptosis by stress-elated survival signals can abrogate the cell cycle arrest andctivate error-prone cell cycle progression thereby generatingutations in the genome of emerged cells [9]. Recently, otherarkers such as p16, �-H2AX, and 53BP1 that are associatedith in vitro senescence have been shown to accumulate with

dvancing age in a number of organisms that include mice [10],nd humans [11] etc. Compelling evidence has indicated that inells approaching senescence, the upstream triggering event ofell cycle inhibitors also contributes to telomere dysfunction [12].hese studies have provided clear molecular evidence that cellsendered senescent by stress, accumulate over time. Since experi-ental evidence for premature senescence induction in the ovarian

pithelium by exogenous agents is scanty, a study involving suchtress-induced response in female reproductive milieu is highlyarranted.

Isocyanates comprise the industrially important group of chem-cals and methyl isocyanate (MIC), one of the highly reactiventermediate, has been shown to cause adverse reproductive healthffects in mammals [13,14]. Previously, we have shown that MICas a genotoxic effect in the ovarian epithelial cells mediatedhrough abnormal stimulation of cell cycle regulators and consid-rable apoptotic response [15]. N-methyl carbamates are a groupf organic compounds which share a common functional groupith the general structure –NH(CO)O– along with isocyanates

nd carbamate esters have alkyl or aryl groups substituted onhe nitrogen, or the amide. Also N-methylcarbamate esters getonverted into isocyanates when the alcohol (–OH) group getsliminated/abstracted. N-succinimidyl N-methylcarbamate is onef the carbamate esters which act as a chemical equivalent tooxic MIC. Based on our current understanding of the molecu-ar biological basis of the effects of isocyanates, we aim at listinghe most important findings that contribute to SIPS of the mam-

alian ovarian epithelium. Also, the characterization of senescencen epithelial cells is still in its beginning. However, a detailedharacterization of senescence in epithelial cells is necessary tonderstand how carcinoma circumvents this program.

. Materials and methods

.1. Reagents and antibodies

N-succinimidyl N-methylcarbamate [CAS No. 18342-66-0] (Sigma Aldrich Lab-ratories, St. Louis, MO, USA) dissolved in 2 mM DMSO with final concentration of.005 �M was used for investigations. The culture petri dishes were procured fromD Falcon (Rockville, MD, USA). Fetal bovine serum was obtained from HyCloneabs (Logan, Utah, USA). Dulbecco’s Modified Eagle’s Medium growth medium wasrocured from Invitrogen Corporation (Carlsbad, CA, USA). Antibiotic–antimycoticolution was obtained from Hi-Media Labs (Mumbai, India). For assessing the DNAamage response proteins Mre11, Rad50, NBS1, cell cycle regulatory proteins p16,21, and pRb and for immuno-FISH analysis of TRF2 protein, antibodies procuredrom Santa-Cruz Biotechnology (Santa Cruz, CA, USA) were used with appropri-te dilutions. DNA cell cycle and ploidy analysis were performed using the BDTM

ycle TEST PLUS DNA Reagent kit, BD Biosciences (San Jose, CA, USA). Cellular senes-ence assay was performed using senescence-associated �-gal (SA-�-gal) stainingit procured from Millipore (Billerica, MA, USA). For assessment of microsatellitenstability, the Wizard® Genomic DNA Purification kit and the PCR II Core Systemit for PCR amplification (Promega Corporation, Madison, WI, USA) were used

.2. Cell lines and culture conditions

B/CMBA.Ov (mouse ovarian epithelial) cells were obtained from the Ameri-

an Type Culture Collection (ATCC; Manassas, VA). The cell line has wild type53 genes without any evident genetic alteration [16]. Cells were seeded athe density of 1 × 105 cells/60 mm culture dishes in DMEM supplemented with0% fetal calf serum and fetal bovine serum, 1% antibiotic–antimycotic (peni-illin/streptomycin/amphotericin), and 2 mM l-glutamine at 37 ◦C in the humidifiedtmosphere of 5% CO2 in air as per ATCC catalogue instructions. After optimum

oxicology 30 (2010) 377–386

confluency, the cells were treated with the experimental agent, N-succinimidyl N-methylcarbamate. At the onset of the experiments, the cells were at an exponentialand asynchronous phase of growth.

2.3. Study design

Experiments were conducted at different time course intervals (n = 3) rangingfrom 6 to 96 h for both controls (untreated) and treated cells. The cells were treatedusing a constant 0.005 �M concentration of N-succinimidyl N-methylcarbamate.The selection of the dose was done on the basis of author’s previous observationsin which this concentration was found optimum to induce DNA damage, oxidativestress and inflammation [17,18].

2.4. DNA damage response

DNA damage kinetics was analyzed qualitatively in terms of ATM/H2AX phos-phorylation through indirect immunofluorescence method. Cells were grownovernight in Dulbecco’s modified eagles medium (DMEM) and subjected to N-succinimidyl N-methylcarbamate treatment for 48 h. The cells were fixed in 10%formaldehyde for 1 h and permeabilized with 0.1% Triton-X- 100 for 30 min, blockedwith 3% BSA for 3 h, and then incubated with anti-ATM/anti-H2AX (dilution 1:1000)for 3 h and FITC/Texas Red conjugated secondary antibodies (dilution 1:200) for 1 h.For qualitative analysis, nuclei were counterstained with DAPI and the cytoskeletonwith phalloidin (phalloidin green for ATM and phalloidin red for H2AX). Prepara-tions were immediately mounted with antifade solution, covered with coverslipsand stored in the dark at 4 ◦C. Images were acquired through the spectral bio-imagingsystem and analyzed with version 4.0 software (Applied Spectral Imaging, EdingenNeckarhausen, Germany).

2.5. Analysis of DNA ploidy

Analysis of DNA ploidy was performed by staining the cultured cells with pro-pidium iodide (PI) according to the manufacturer’s protocol. PI fluorescence wasmeasured with BDTM FACS Calibur 6.0. A sum total of 30,000 events was acquiredand subjected for analysis by ModFit LT 3.0 (Verity Software House Inc., Topsham,ME, USA). Histogram displays were overlaid with graphical representations of themodeled G0/G1, S and G2/M populations following ModFit-analysis and data wereexpressed as percentages of cells for any given phase of the cell cycle.

2.6. Western blot analysis

Briefly, cells were rinsed twice in PBS and lysed in the buffer (10% SDS, 1 MTris pH-7.6, 5 mM EDTA). The obtained cell lysates were centrifuged at 12,000 rpmfor 10 min at 4 ◦C, and the supernatant was collected. Protein concentrationswere determined from the supernatant by Bradford assay. An amount of 100 �gprotein was analyzed through 10% sodium dodecyl sulfate polyacrylamide gel elec-trophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane in 25 mMTris, 194 mM glycine, and 20% methanol at 4 ◦C with semi-dryer transfer unit of Hoe-fer (Holliston, MA, USA). Membranes were blocked with 5% non-fat milk in 0.1% PBSTand incubated for overnight at 4 ◦C with primary mouse monoclonal antibodies spe-cific for GADD45/Mre11/Rad50/Nbs1/p21/p16/pRb (1:1000 dilution). Membraneswere washed three times for 20 min with 0.1% PBST and incubated for 2 h at roomtemperature with horse radish peroxidase conjugated secondary antibody (1:2500dilutions). Membranes were washed, and bound antibodies were analyzed throughECL Plus (Amersham Biosciences, Pittsburgh, PA, USA). Quantitation of signals wasperformed by using QuantiOne software (Bio-Rad, Philadelphia, PA, USA).

2.7. Senescence

SA-�-gal activity is commonly used as a senescent marker. Since this enzymeworks under acidic pH conditions in a senescence-specific manner, senescent cellscan be recognized as positive cell by this assay. Cellular senescence assays wereperformed through senescence-associated �-gal staining (SA-�-gal) in compliancewith the manufacturer’s instruction manual and stained cells were examined andphotographed under phase contrast microscope.

2.8. Cellular transformation assay

The ability of cells to grow in the absence of adhesion is one of indicators forassessing cell transformation. Briefly, a 1% agarose solution was prepared in dou-ble distilled water and heated in microwave oven until agarose is melted and wasallowed to cool for 5–10 min (with the cap partially screwed on). A volume of 5 ml

of agarose was added to a 10 cm2 petri dish and rotated until the plate is completelycovered and the agarose was pipetted off leaving a thin film of agarose on the bot-tom of the petri dish). Plates were dried for 20 min with lid on. Cells with density of2 × 106 in 10 ml of culture media were plated and monitored for colony formationof cells daily with regular replenishment of media. Microphotographs of colonieswere taken under phase contrast microscope as and when needed.

G.V. Raghuram et al. / Reproductive Toxicology 30 (2010) 377–386 379

Fig. 1. Qualitative analysis of DNA damage. (a) Microphotographs (200×) showing immunofluorescence analysis of ATM phosphorylation with significant punctuated nuclearpatterns in B/CMBA.Ov (mouse ovarian epithelial) cells following treatment with 0.005 �M N-succinimidyl N-methylcarbamate at 48 h (lower panel) in comparison to controls(upper panel). (b) Microphotographs (200×) showing immunofluorescence analysis of H2AX phosphorylation with distinct interspersed pattern of �-H2AX foci in nucleus ofB (lowec n) forl pretatv

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/CMBA.Ov cells treated with 0.005 �M N-succinimidyl N-methylcarbamate at 48 hounterstained with DAPI staining while cytoskeleton stained with phalloidin (greeabeled in Texas red while �-H2AX foci have been stained in FITC (green). (For interersion of this article.)

.9. Cytogenetic analysis

.9.1. Structural and numerical chromosomal aberrationsBriefly, exponentially growing (50–80% confluence) B/CMBA.Ov cells were

xposed to 0.04 �g/ml colchicine (Sigma Aldrich Laboratories, St. Louis, MO, USA) forh at 37 ◦C. Metaphase spreads were prepared and stained by conventional meth-ds as described previously [19]. A minimum of 50 metaphases for control andreated cells was selected and photographed, analyzed, to determine chromosomerequency distribution and morphology.

.9.2. Immuno-FISH of TRF2Briefly, metaphase spreads from both control and treated cell suspension in

resh Carnoy’s fixative were prepared and left for ageing overnight at RT. The cellsere fixed in 4% formaldehyde for 1 h and permeabilized with 0.1% Triton-X-100

or 30 min, blocked with 3% bovine serum albumin (BSA) for 1 h, and then incubatedith anti-TRF2 antibody (dilution 1:1000) for 2 h and FITC-conjugated secondary

r panel) in comparison to controls (upper panel). Blue color represents the nucleusATM/phalloidin (red) for H2AX. The signals of the ATM phosphorylation have beenion of the references to color in this figure legend, the reader is referred to the web

antibody (dilution 1:200) for 1 h. Nuclear counterstaining with DAPI was performedand immediately mounted with antifade solution, cover slipped and stored in thedark at 4 ◦C. The images were acquired and analyzed as mentioned above.

2.10. Microsatellite instability

Assessment of microsatellite instability by intersimple sequence repeat PCRwas done by extracting genomic DNA using the supplier’s protocol and 50 ng ofthe extracted DNA was amplified in a 10 �l reaction mixture containing 1X PCRbuffer, 200 �M deoxynucleoside triphosphates, 1.5 mM MgCl2, 2.5 �M of one of the

base-anchored dinucleotide repeat (CA)8 primers, 0.5 units Taq DNA polymerase.The cycling conditions were 3 min initial denaturation at 94 ◦C, 30 cycles of 30 s at94 ◦C, 45 s at 50 ◦C, and 2 min at 72 ◦C, followed by a final extension for 7 min at 72 ◦C.Genomic alterations were detected as gains and losses of specific bands followinganalysis of the PCR product on 8% non-denaturing polyacrylamide gels containing5% glycerol and visualization under a gel documentation system.

380 G.V. Raghuram et al. / Reproductive Toxicology 30 (2010) 377–386

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ig. 2. Status of stress responsive growth arrest proteins. (a) Western blot analysollowing treatment with 0.005 �M N-succinimidyl N-methylcarbamate at differen-actin as loading control. The blots represent one of three reproducible experimenrotein compared with parallel controls. The cells were observed from 6 to 72 h an

.11. Statistical analysis

Student’s t-test was employed for statistical comparisons and data are expresseds mean ± SEM. P values ≤0.05 were considered as statistically significant.

. Results

.1. DNA damage response and repair

Immunofluorescence analysis revealed persistence of DNAamage signaling factors ATM/�-H2AX through amplification inheir phosphorylation states at Ser1981/Ser139 in a punctuatednd interspersed nuclear pattern even after treatment for 48 hn comparison to respective controls (Fig. 1a and b). At the

ig. 3. Status of DNA repairs protein. (a) Western blot analysis of DNA repair proteins Mre1-methylcarbamate at 06, 12, 24, 48, 72 h in B/CMBA.Ov cells. Blots were probed with �-axpression of Mre11, Rad50 and Nbs1 proteins increased in time dependent manner withxpression) for Mre11, Rad50 and Nbs1 proteins compared with parallel controls. The control.*P ≤ 0.001 and **P ≤ 0.05.

wing expression of DNA damage responsive GADD45 protein in B/CMBA.Ov cellsintervals starting from 6 to 72 h in contrast to control cells. Blots were probed with) Histogram showing relative band density (in percentage expression) for GADD45

were probed with �-actin as loading control.*P ≤ 0.001.

same time, western blot analysis of DNA damage responsivegrowth arrest protein GADD45 revealed increased accumulationof its levels along the time course till 72 h in contrast to theparallel controls as indicated by the increasing intensity of band-ing pattern (Fig. 2a). Software band density analysis GADD45protein in treated cells also showed incremental percentage ofexpression from 6 to 72 h (P ≤ 0.001) in comparison to paral-lel controls (Fig. 2b). Simultaneously, analysis of damage repair

factors Mre11, Rad50 and Nbs1 yielded similar augmentation intheir expression pattern in a time dependent manner respec-tively in contrast to their controls (P ≤ 0.05, P ≤ 0.001) (Fig. 3)thereby implicating the severity of stress-induced DNA dam-age.

1, Rad50, Nbs1 in control (C) and following treatment with 0.005 �M N-succinimidylctin as loading control. The blots represent one of three reproducible experiments.

respect to control. (b)–(d) Histograms showing relative band density (in percentageells were observed from 6 to 72 h and blots were probed with �-actin as loading

G.V. Raghuram et al. / Reproductive Toxicology 30 (2010) 377–386 381

Fig. 4. DNA ploidy analysis through flow cytometry in cells prior to and following treatment with 0.005 �M N-succinimidyl N-methylcarbamate (a) Control showingpercentage of cells in G1(red), S (striated blue) and G2/M (red) phases of the cell cycle respectively, (b) Cells after 12 h treatment showing significant arrest in G1 phaseand apoptotic (Apo, blue peak) phase (c) a distinct sub-G1 peak showing onset of aneuploidy (Aneu, yellow peak) in cells after 48 h treatment, (d) cells after 96 h treatmentshowing significant increase in aneuploidy with considerable G1 arrest. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)

Fig. 5. Status of cell cycle inhibitors. (a) Immuno-blot analysis displayed immediate and temporal up-regulation in CDK inhibitors (p21, p16, pRB) in B/CMBA.Ov cellsexposed to 0.005 �M N-succinimidyl N-methylcarbamate at indicated time intervals in comparison to unexposed (control) cells. The blots represent one of three reproducibleexperiments and were probed with �-actin as loading control. (b)–(d) Histograms showing relative band density (in percentage expression) for p21, p16 and pRb proteinscompared with parallel controls. The cells were observed from 6 to 72 h and blots were probed with �-actin as loading control. *P ≤ 0.001 and **P ≤ 0.05.

382 G.V. Raghuram et al. / Reproductive Toxicology 30 (2010) 377–386

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ig. 6. Senescence-associated-�-gal activity and neoplastic transformation. (a) Gra-succinimidyl N-methylcarbamate treated cells along the time course of 12–72 h.nd after treatment with 0.005 �M N-succinimidyl N-methylcarbamate at, 24 h, 48 htain at 48 and 72 h (small white arrows), transformed phenotype at 96 h (big white

.2. Analysis of DNA ploidy

Treated cells showed early halt in cell cycle progression at 12nd 48 h through G1 check point activation (Fig. 4). Interestingly,neuploid cells were observed at 48 h which further increased after6 h of exposure with a distinct yellow peak suggesting pertur-ation in ploidy levels due to N-succinimidyl N-methylcarbamatereatment.

.3. Cell cycle regulation

We observed an immediate up-regulation of p21WAF/Cip1 asarly as 6 h after the treatment with significant percent increase

n expression by 96.5 ± 1.2 (P ≤ 0.001) as compared to control cellshowing 46.1 ± 0.9 (Fig. 5a and b). This increase was evident whenells were bound to accumulate in G1 phase of cell cycle (Fig. 4)efore and after 12 h of treatment with 0.005 �M of MIC substitute

n comparison to control cells (P ≤ 0.001). Also, there was subse-

owing percent �-gal positive staining in B/CMBA.Ov cells in control and 0.005 �Mpresentative phase contrast microphotographs (200×) of B/CMBA.Ov cells prior to, 96 h and 8 days. Note the treated cells showing increased in accumulation of �-gal) and anchorage-independent neoplastic growth after 8 days of culture in soft agar.

quent, but, temporal upshot of p16INK4A and pRb levels after 12 and48 h of treatment in comparison to control cells (Fig. 5a). Quantita-tive evaluation of p16INK4A band intensity yielded high percentageof expression temporally at 12 h (P ≤ 0.001) as opposed to parallelcontrol (Fig. 5c), while pRb bands presented the sequential increasein expression pattern at 48 h (P ≤ 0.001) in comparison to its parallelcontrol by (Fig. 5d). These results reinforced their role as senescencemarkers.

3.4. SA ˇ-gal Activity

N-succinimidyl N-methylcarbamate caused a gradual increase

in SA-�-gal activity over time, as judged by cytochemicalstaining. Presenescent B/CMBA.Ov cells treated with 0.005 �M N-succinimidyl N-methylcarbamate for 24 h displayed a perceptibleincrease in SA-�-gal staining (Fig. 6a), whereas untreated cellsgrown in parallel were negative. The majority of cells treated

G.V. Raghuram et al. / Reproductive Toxicology 30 (2010) 377–386 383

Fig. 7. Flow cytometric measurement of cell size. Histogram representing thechanges in cell size induced by 0.005 �M N-succinimidyl N-methylcarbamate inB/CMBA.Ov cells in comparison to control. The representative curves correspond to((c

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Fig. 8. (a) Cumulative structural chromosomal aberrations in B/CMBA.Ov cells fol-lowing exposure to constant 0.005 �M dose of N-succinimidyl N-methylcarbamateacross time intervals of 6, 24, 48, 72, 96 h. Graph representing frequency (%) of totalchromosome structural aberrations (CSA) per cell observed in treated cells in com-parison to control. Total CSA considered were centric fusions, chromatid breaks,ring, dicentrics, chromosomal fragments, and chromosomal end-to-end fusions.(b) Numerical imbalance caused in metaphase spreads after exposure to 0.005 �MN-succinimidyl N-methylcarbamate. Histogram depicting percent gain and loss ofchromosomal numbers in B/CMBA.Ov cells at different time points 6, 24, 48, 96 hof incubation. (c) Telomeric localization of TRF2 protein. Expression signals of TRF2at the ends of metaphase chromosomes stained with the goat anti-rabbit antibody(FITC green) from control cells, while treated cells showing comparatively less or

I) control untreated cells at 0 h and cells observed during 24 h (II), 48 h (III) and 72 hIV) of treatment. The shift to the right of the curve implicates an increase in theellular size during acquisition of senescence phenotype along the time course.

ith MIC substitute displayed light staining after 24 h, moderatetaining after 48 h, and intense staining by 72 h unveiling a typi-al senescence-specific morphology with ‘flattened and enlarged’hape as early as at 24 h (Fig. 6b). Besides, the cell size of N-uccinimidyl N-methylcarbamate treated cells, as measured byow cytometry analysis, was altered leading to expansion of theirize with the duration of exposure as opposed to the control onesFig. 7). The rise in production of a blue-dyed precipitate that resultsrom the cleavage of the chromogenic substrate X-Gal by theseells is indicative of accumulation of lipofuscein granules and theremature senescence-associated �-galactosidase activity underxtrinsic stress conditions.

.5. Cytogenetic analysis

Cytogenetic analysis of B/CMBA.Ov cells treated with N-uccinimidyl N-methylcarbamate revealed interesting results. Webserved an increase in total chromosomal structural aberrationser cell involving chromatid fragmentation (fr), centric fusionscf), dicentrics (dic), chromatid breaks (cb), ring (r) and end-to-nd fusion (eef) at different time intervals 06, 12, 24, 48 and2 h of treatment. As the duration of the exposure increased, theean incidence of percent aberration rate per cell also elevatedith values of 0.53 ± 0.03 at 6 h to 3.26 ± 0.06 at 72 h respectively

P < 0.001; Fig. 8a). Simultaneously, evaluation of the numericalytogenetic index revealed a significant percent gain and loss ofhole chromosomal number as early as 24 and 48 h of treatment

Fig. 8b).

.6. Analysis for TRF2

Immuno-FISH study on metaphase spreads of B/CMBA.Ov cellsreated with N-succinimidyl N-methylcarbamate demonstratedoss of TRF2 protein expression near the telomeric regions. Notably,he study also revealed the chromosomal end fusions which are

anifested as a result of deficiency of TRF2 protein levels therebyending evidence to the induction of stress-induced prematureenescence (Fig. 8c).

.7. Microsatellite instability

Microsatellite repeat DNA of treated cells upon ISSR PCR analy-is presented variation in the amplification of intersimple sequenceepeats (CA)8 to the fore at 72 h itself. Thus, deviation in the

no signals of TRF2 at telomeres (small white arrow) and big white arrow highlightno TRF2 signals on centric fusion. DNA was counterstained with DAPI (blue). (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

both presence or absence and increased or decreased intensityof bands portrayed the occurrence of microsatellite instability(Fig. 9).

3.8. Neoplastic transformation

Ability of cells to grow in the absence of adhesion is one of thehallmarks of transformation and evaluation of the soft-agar colonyformation assay in N-succinimidyl N-methyl carbamate treated

384 G.V. Raghuram et al. / Reproductive T

Fig. 9. Microsatellite instability. Representative intersimple sequence repeat (ISSR)PCR fingerprints displaying the fluctuating banding pattern of microsatelliterepeats (CA)8 generated from the genomic DNA of 0.005 �M N-succinimidyl N-mmwr

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ethylcarbamate-exposed B/CMBA.Ov cells and control cells. Lane 1: low base pairarker (100 bp); Lane 2: control; Lanes 3 and 4: treated cells at 48 and 96 h. Note: bighite arrow showing gains and small white arrows indicating loss of microsatellite

epeats.

ells showed colonies of transformed cells as early as 8 days impli-ating anchorage-independent growth (Fig. 6b).

. Discussion

During the past decades there has been substantial focus on thempact of environment on reproductive health. Toxic chemicals areited as agents that may be linked to negative reproductive healthutcomes through their deleterious effects on ovarian function. Iso-yanates such as MIC though industrially indispensable, have beenroven to show adverse health effects in this regard by inducingNA adducts [15]. Since OSE is critical for primary ovarian functionnd tissue repair, any defects in ovarian epithelium could underlieith reduced fertility. However, molecular mechanisms underly-

ng their role in stress-induced premature senescence in epithelialells have not received much attention than needed.

Recent findings have experimentally linked the DNA damagend organismal aging with fact that longevity-regulating geneticathways respond to the accumulation of DNA damage alongith other stress conditions and conversely influence the rate ofamage accumulation [20]. The current study demonstrated thishenomenon through increased nuclear retention of phospho-

TM/�-H2AX foci in N-succinimidyl N-methyl carbamate treatedells even after 48 h of exposure (Fig. 1a and b). Impaired DNAamage repair, have been demonstrated to inclination towards seg-ental progeroid syndromes in mammals [21]. The integrity ofNA of surface epithelial cells circumjacent to the ovarian rupture

oxicology 30 (2010) 377–386

site is compromised during the ovulatory process making themapparently sensitive to genotoxins [22]. GADD45 family mem-bers are rapidly induced by genotoxic stress agents and previousstudies have demonstrated that GADD45 genes play an impor-tant role in senescence [23,24]. In the present study, we observedthe altered expression of GADD45 protein in treated cells allalong the time course (Fig. 2). Moreover, deregulated DNA dam-age repair, especially due to inter-strand DNA cross-link (ICL)has been suggested to contribute to accelerated aging in mam-mals [25]. It has been evidenced that GADD45 expression has afunctional role in DNA excision repair [26]. DNA double-strandbreak (DSB) signaling in response to ICL agents has been identi-fied as a key inducer of cellular senescence through alterationsin the recruitment and assembly of Mre11, Rad50, Nbs1 (MRN)complex proteins [27]. Our study here extends this propositionthrough improper recruitment of Mre11/Nbs1/Rad50 proteins inN-succinimidyl N-methylcarbamate treated cells over time (Fig. 3)causing an escalating loss of function with an inability to main-tain replicative fidelity of the genome. We postulate that increasedaccumulation of these repair factors might be in response to thestress stimulus induced by MIC substitute.

In addition, excess accumulation of DNA damage responsivefactors cause early cell cycle arrest. It has been shown that cellcycle-related proteins regulate the proliferation and differentia-tion and morphological appearance of ovarian surface epithelium[28]. Generally, the cell cycle exit associated with differentiationis accompanied by the induction of senescence program, and inthis course, certain molecular signals such as CDK inhibitors (Rb,p16INK4A and p21WAF/Cip1play critical roles in mediating DNAdamage-induced senescence through their up-regulation [29,30].Concurrently, in the present investigation the N-succinimidyl N-methylcarbamate treated cells showed an immediate and temporalup-regulation of p21WAF/Cip1 p16INK4a and pRb after 12 h and 48 h oftreatment (Fig. 5) in contrast to respective controls before the emer-gence of SA-�-Gal positive cells. These results further validatedtheir hallmark feature in the untimed initiation of stress-associatedsenescence.

Meanwhile, up-regulation of CDKIs has been speculated to influ-ence cell enlargement, activation of the SA-�-gal activity. Inductionof a senescence-like phenotype also occurs after environmentaland genotoxic insults [31,32] and several treatments can stimu-late and accelerate a senescence-like state prematurely in youngcells [33,34]. Consistent with this, in the present investigation, weobserved an increase in the cell size, a characteristic feature ofsenescence, in response to exposure to MIC substitute right from 6 honwards to 24 h (Fig. 7). It has been suggested that the accumulationof senescent cells within ovaries might play a role in the develop-ment of ovarian diseases [35]. Data presented here display the earlyinduction of positive staining for �-galactosidase in MIC substituteexposed cells in-advance from the 24 h post treatment itself withlarge granular nucleus and focal enrichment of lysosome-related�-galactosidase activity at vacuoles which sustained progressivelytill 72 h (Fig. 6a and b). These outcomes further support the notionthat an increase in lysosomal mass is responsible for the increasein �-galactosidase activity observed in senescent cells [36].

Moreover, DNA damage checkpoint activation is postulated tobe causally associated with the senescent state when cells senseunrepairable and extensive chromosomal abnormalities and as aresult the telomeric ends are known to directly participate in theDNA damage response [37]. It was pointed out that any defectsin DNA damage response pathways might also result in chromoso-

mal defects and/or premature cell senescence in culture, raising thepossibility that these might initiate various phenotypes associatedwith chromosomal fragility syndromes [38]. Additionally, it is wellknown that increase in rate of gross chromosomal rearrangementsfrequently accelerate aging [39] and specifically in premature age-

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ng, autosomal chromosome aberrations have been very commonlyncountered in senescent cells [40]. Recent data have indicatedhat extrinsic cellular stresses in mammalian ovarian epithelialells through genotoxic chemicals exposure can cause chromoso-al aberrations [41,42]. Incidentally, our present cytogenetic study

evealed the early presentation of chromosomal imbalance at 24 hith successive increment in gross chromosomal abnormalities

core and significant proportion of gain and loss of whole chro-osomes along duration of exposure (Fig. 8a and b). These data

ndicate that N-succinimidyl N-methylcarbamate exposure leadso spontaneous chromosomal abnormalities that accumulate overime and are associated with premature cellular senescence. Fur-hermore, these results along with significant alterations in ploidyevels observed after 96 h (Fig. 4) provides possible link whichllustrates that premature senescence can contribute to defectiveenome maintenance program.

Genome maintenance, in particular, maintenance of telom-re function is critical to ensure longevity needed for replicationnd as a consequence imperfections or defects in maintaining theenome might lead to abnormal cell proliferation [43]. Dysfunctionf telomeres is sufficient to produce severe affect on proper func-ion of telomere-associated protein resulting in premature tissueegeneration [44]. The telomere-associated protein TRF2 (telom-ric repeat binding factor 2) is critical for the control of telomeretructure and function. Previous findings suggest that telomerettrition can accelerate senescence in mitotic cells and dysfunc-ion of TRF2 results in the exposure of the telomere ends andctivation of ATM (ataxia telangiectasia mutated)-mediated DNAamage response [45,46] to repair the chromosome ends, whichould manifest in chromosomal end joining and lead to dev-

stating consequences for genomic integrity after stress stimuli47]. In the present immuno-FISH study, we witnessed the lossf TRF2 expression at the chromosomal ends of treated cells at8 h (Fig. 8c), thereby, fostering our cytogenetic observations thatelomeric ends are susceptible to the N-succinimidyl N-methyl car-amate exposure and resultant initiation of premature senescenceue to inappropriate activation of DNA repair factors as evidencedy our above mentioned western blot results.

Senescence-related accumulation of mutations has been exten-ively documented, and it has been proposed as one of therominent causes of age-associated genomic instability. Previoustudies have shown that as in vitro cell senescence advances,icrosatellite instability also develops progressively [48]. In the

resent study, we observed the commencement of microsatellitenstability in N-succinimidyl N-methylcarbamate exposed cells at8 h which later on progressed with loss of (CA)8 repeats at 96 hFig. 9). On the other hand, in some cells, senescence-associatedNA damages could statistically affect a cocktail of oncogenes and

umor-suppressor genes favoring an evolution towards transfor-ation or immortalization as illustrated in the premature aging

henotype [49]. Consistent with this in the current study, webserved morphological variations in treated cells at 96 h indicatingmergence of cells with overcoming of senescence (Fig. 6b).

In order for transformed cells to be generated from senescentells, there must be some mutagenic events that occur duringenescence. The importance of a high rate of mutations associatedith senescence in the development of tumors is highlighted byany progeria syndromes where aging occurs prematurely and

s associated with numerous DNA alterations and mutations andncreased cancer incidence [50]. Concurrently, in our study, in vitroellular transformation assay after 8 days of culture presented the

triking appearance of anchorage-independent growth of treatedells in clusters having refractile property along with a signifi-ant loss of SA-�-gal activity (Fig. 6b) suggesting that cells, alreadyartially transformed, are highly prone to further neoplastic devel-pment upon exposure to exogenous genotoxic agents.

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oxicology 30 (2010) 377–386 385

In conclusion, isocyanates in the form of N-succinimidyl N-methyl carbamate induce stress-induced premature senescencethrough persistent activation of DNA damage response, abruptup-regulation of cell cycle inhibitors with early and increasedexpression of SA-�-gal activity. Besides, our study also hintstowards the plausible association of premature senescence withgenomic instability through significant alterations in chromoso-mal structure and number. In addition, these findings suggest thatsenescent cells could evolve towards tumor-initiated cells throughformation of pre-neoplastic lesions. Further characterization ofthe molecular pathways involved and genes regulated in the OSEwould not only contribute to a more detailed understanding ofthe ovarian epithelium transcriptome, but also ultimately lead toa better understanding of the aberrations leading to transforma-tion in the ovarian epithelium. Translating the knowledge gainedon premature exogenous stress-induced senescence into identi-fying the putative factors and understanding the mechanisms ofovarian disorders would be of the greatest pursuit to the scientificcommunity.

Conflict of interest statement

None declared.

Acknowledgements

The authors are thankful to Mr. Naveen Kumar Khare for pro-viding necessary technical assistance.

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