Organochlorine pesticides induce inflammation, ROS production, and DNA damage in human epithelial ovary cells: An in vitro study

Chemosphere 246 (2020) 125691

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Organochlorine pesticides induce inflammation, ROS production, and DNA damage in human epithelial ovary cells: An in vitro study Harendra Kumar Shah, Tusha Sharma, Basu Dev Banerjee* Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (University of Delhi) & GTB Hospital, Dilshad Garden, Delhi, 110095, India

h i g h l i g h t s
OCPs (b-HCH, DDE, and Dieldrin) exposure revealed inflammatory response in Human Ovary Surface Epithelial (HOSE) cells.
ROS generation significantly higher in HOSE cells treated with OCPs.
Inflammatory cytokine modulators such as NF-kB, and COX-2 significantly higher in HOSE cells exposed to OCPs.
Overexpression of pro-inflammatory cytokines such as IL-6, IL-1b and TNF-a in OCPs exposed HOSE cells.
Significant DNA damage in HOSE cells following exposure to OCPs.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 July 2019 Received in revised form 15 December 2019 Accepted 16 December 2019 Available online 19 December 2019

Although the etiology of ovarian cancer is not clear, certain factors are implicated in this disease, such as ovulation, gonadotropic and steroid hormones, growth factors, cytokines, environmental agents, etc. Epidemiological studies have proven environmental exposure to pesticides with an increased risk of Epithelial Ovarian Cancer (EOC); however, the molecular mechanism underlying the carcinogenic effects of pesticides in human ovary remains poorly understood. The present study aimed to study the proinflammatory response of organochlorine pesticides (OCPs) namely b-hexachlorocyclohexane (b-HCH), dichlorodiphenyldichloroethylene (DDE) and Dieldrin following exposure to human ovary surface epithelial cells (HOSE) for risk prediction of epithelial ovarian cancer. We found high level of Reactive oxygen species (ROS) production and DNA damage along with up-regulation of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6, nuclear factor kappa B (NF-kB) and cyclooxygenase (COX)-2 expression in OCPs treated HOSE cells compared to control (DMSO). The result of the present study suggests that b-HCH, DDE, and Dieldrin exposure induce ROS and proinflammatory response as well as DNA damage in HOSE cells. These various results show that OCPs may account for the neoplastic transformation of HOSE cells in the ovary. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: A. Gies Keywords: Organochlorine pesticides Human ovary surface epithelial cell Inflammation Cytokine DNA damage ROS Epithelial ovary cancer

1. Introduction Organochlorine pesticides (OCPs) such as hexachlorocyclohexane (HCH), DDT, and Dieldrin are among the most widely used as insecticides (Gupta, 2004; Lallas, 2001). OCPs like

Abbreviations: OCPs, organochlorine pesticides; b-HCH, b-hexachlorocyclohexane; DDE, dichlorodiphenyldichloroethylene; HOSE, human ovary surface epithelial; DMSO, dimethyl sulfoxide; ROS, Reactive oxygen species; FBS, fetal bovine serum; EOC, Epithelial Ovary Cancer. * Corresponding author. University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi, 110095, India. E-mail address: [email protected] (B.D. Banerjee). https://doi.org/10.1016/j.chemosphere.2019.125691 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

DDT and Dieldrin are persistent organic pollutants that may accumulate in the environment and food (Malarvannan et al., 2009). It is estimated that >90% of applied insecticides and herbicides never reach to their target instead they remain persistent for a longer period of time (even in years) in the environment and contaminate air, water, and food making them very likely to affect non-target organisms including humans (Safe, 1994). As a result, human get persistent exposure to these pesticides and become more susceptible to various pathological condition including cancer such as Hodgkin & non-Hodgkin lymphoma, melanoma, multiple myeloma, leukemia, breast cancer, ovarian cancer, prostate cancer, lung cancer, brain cancer, colorectal cancer, testicular cancer, pancreatic cancer, esophageal cancer, stomach cancer, and skin

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cancer (Alavanja and Bonner, 2012; Kumar et al., 2010; Mathur et al., 2008; Mnif et al., 2011; Shah et al., 2018; Sharma et al., 2015; Sharma et al., 2013; Snedeker, 2001; Stoytcheva, 2011; Weichenthal et al., 2010; Zhang et al., 2014). Based on various in vitro, in vivo and epidemiological finding EPA’s Pesticides program suggested 70 pesticides as possible carcinogens including bHCH, DDT and Dieldrin (Tiemann, 2008; Turusov et al., 2002; Zucchini-Pascal et al., 2012). The Stockholm Convention listed the first 9 pesticides including Dieldrin, DDT and later b-HCH which have recognized as causing various adverse effects on human health and the ecosystem. Inflammation is an immediate and essential host defense in response to infection or tissue injury. It is estimated that 15% of human cancers are associated with chronic inflammation (He and Karin, 2011). Persistence of inflammation for a longer period results in chronic inflammation that may predispose the host to various chronic illnesses, including cancer (Lin and Karin, 2007). The sustained release of pro-inflammatory ROS, cytokines, as well as Cyclooxygenase (COX)-2, potentiate genomic instability as well as induce various transcription factors, including nuclear factor kappa B (NF-kB) which have shown to play a crucial role in tumorigenesis (Federico et al., 2007). Pesticides can activate innate immune dysfunction leading to chronic inflammation results in increased secretion of inflammatory cytokines (TNF-a, IL-1, 6 and 8) facilitate tumor cell growth (Grivennikov and Karin, 2011; Mokarizadeh et al., 2015). Pesticide-induced oxidative stress may promote carcinogenic mutations via induction of DNA damage (Tebourbi et al., 2011). Ovarian cancer is the second most common and leading lethal type of gynecological cancer in women (Siegel et al., 2019) and can originate in the germ cells, sex-cord stroma, fallopian tube (FT), or ovary epithelium. Epithelial ovarian cancer (EOC) originates from the ovary and fallopian tube epithelium is the most common histological type of ovarian cancer and accounts for 85e90% among all ovarian cancer (Wei et al., 2013). Many theories of ovarian cancer have been proposed, including incessant ovulation, increased cellular senescence and uncontrolled production of reactive oxygen species (Fleming et al., 2006; Saed et al., 2017). The exact cause of ovarian cancer remains unclear; however, a number of associated risk factors have been identified such as perimenopause, postmenopause, age, hormone, hereditary, lifestyle, inflammation, exposure of the environmental factors such as talc and asbestos, etc to the genital organ (Salehi et al., 2008). Moreover, the recent studies from our lab have shown the association of OCP in epithelial ovarian cancer patients, however, the exact cellular and molecular mechanism underlying carcinogenic effects of OCPs in human ovary epithelial cells remain poorly understood. Therefore in the present study, we hypothesized that persistence exposure to OCP residues may induce chronic inflammation to the human ovary surface epithelial cells (HOSE) cells by dysregulating cytokine and cytokine modulators in the tumorigenesis of epithelial ovarian cancer. Hence, we aim to study the impact of OCPs such as b-HCH, DDE, and Dieldrin exposure to HOSE cells in the pro-inflammatory responses, one of the crucial pathways in the process of tumorigenesis. 2. Materials and methods 2.1. Materials Human ovary surface epithelial (HOSE) cells at passage one, ovarian epithelial cell medium (OEpiCM), growth supplement and Penicillin/streptomycin solutions was obtained from Science cell (Germany). MTT reagent and DMSO were obtained from Himedia, India. OCPs (b-HCH, DDE, Dieldrin), and 20 , 70 -

dichlorodihydrofluorescein diacetate (H2DCFDA) were obtained from Sigma-Aldrich. Trizol reagent was supplied by Invitrogen (Carlsbad, CA, USA). The plasticware was obtained from Nunc (Thermo Scientific, USA). 2.2. Cell culture HOSE cells were cultured in a humidified atmosphere of 5% CO2 at 37  C in ovarian epithelial cell medium (OEpiCM) supplemented with growth factors and penicillin/streptomycin solution. When the cell culture reached 50% confluence, cells were treated with different OCPs such as b-HCH, DDE and Dieldrin. All the experiments were done within the 5th passage as per the manufacturer’s instruction; the cells are guaranteed to further expand for five populations doubling without cell modification. 2.3. Pesticide treatment HOSE cells were treated with various concentration of b-HCH, DDE and Dieldrin (1, 10, 20, 30, 40, 50, 100 and 200 mM). Before treatment, all the pesticides were solubilized in a 100% DMSO solution, then diluted in the OEpiCM medium to reach the final DMSO concentration less than 0.25% (which had been previously proven not to be cytotoxic for the cells). Treatments with OCPs were renewed every 48 h for 7 days. Control cells were exposed to 0.25% DMSO alone. 2.4. Evaluation of cell viability/cytotoxicity The impact of OCPs on HOSE cell viability was determined by measuring the conversion of tetrazolium salt 3-(4, 5dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide (MTT), to formazan, as previously described (Mosmann, 1983). Briefly, HOSE cells were seeded in 96-well plates (104 cells/well). At 50% confluency, the cells were treated with various concentration of bHCH, DDE and Dieldrin (1, 10, 20, 30, 40, 50, 100 and 200 mM) during 3 and 7 days. The culture medium was then discarded and the monolayer washed twice with PBS. The cells were then incubated with MTT (20 ml/well at a concentration of 5 mg/ml) in OEpiCM media for 2 h at 37  C. Supernatant was discarded and 200 ml of DMSO was added to dissolve the formazan crystal. The absorbance of formed formazan solution was measured by a MultiMode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 570 and 690 nm. The relative cell viability was expressed as the ratio (%) of the absorbance in the experimental wells to that of the control wells (cells treated with DMSO). Based on calculated IC50 value by cytotoxicity analysis and the concentrations commonly used for in vitro studies in the literature, we have selected the highest 20 mM concentration of b-HCH, DDE and Dieldrin in the present study. All experiments repeated independently 3 times in triplicate. 2.5. Observation of morphologic changes HOSE cells were seeded in 6 well plates at density 1.25  105 cells/well and treated at 50% confluence with 20 mM concentration of b-HCH, DDE and Dieldrin for 7 days and then morphological changes were analyzed using inverted microscope (EVOS XL CORE, Life Technologies). 2.6. Measurements of reactive oxygen species (ROS) levels in the cells Intracellular ROS generation was assessed by using 20 , 70 dichlorodihydrofluorescein diacetate (H2DCFDA), Sigma after

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exposure of 20 mM b-HCH, DDE and Dieldrin for 3 days. HOSE cells seeded into 96 well plates at a density of 104 cells/well in OEpiCM media. Then cells were incubated with OCPs and 0.1% DMSO (control) for 3 days. For positive control, the cells were treated with H2O2 (50 mM) for 1hr before adding H2DCFDA. Then, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated with 50 mM H2DCFDA (the stock solution was made in DMSO, and further diluted in HBSS so that the final concentration in the medium was 0.1%) for 30 min in CO2 incubator. After washing twice with ice-cold PBS, the formation of ROS was determined immediately at excitation and emission wavelengths of 485 nm and 535 nm, respectively, after subtracting the background. Percentage of ROS are measured as %OCP treated cells/% control (DMSO)*100.

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2.8. Agarose gel electrophoresis Thus obtained amplified product from real-time RT PCR was further validated by running on 1.6% agarose gel to confirm the actual length and band intensity.

2.9. ELISA The levels of secreted IL-6 were estimated from culture soup of HOSE cells following 7 days of exposure OCPs and control cells (DMSO) using commercial available RayBio Human IL-6 ELISA kit (Norcross, GA) according to the manufacturer’s instructions. Kit sensitivity was <3 pg IL-6/ml and the average % of recovery is 95.65 in cell culture media.

2.7. RNA isolation and real-time RT-PCR Total RNA was extracted from control and OCPs treated HOSE cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) (Schmittgen and Livak, 2008). The yield and quality of the extracted RNA were estimated using Nanodrop (Thermo Scientific, Waltham, MA, USA). One microgram of total RNA was reverse transcribed using a Verso cDNA synthesis kit (Thermo-Fischer Scientific) as per manufacturer’s description. Real-time RT-PCR analysis was carried out with CFX Connect Real-Time System (Bio-Rad, USA) following the MIQE Guidelines. A total reaction volume of 20 mL, containing 10 mL SSo-Fast™ Eva green® supermix (Bio-Rad, USA), 1 mL (10 rmol) of each forward and reverse primer, 1 mL (1 mg) cDNA and 7 mL of nuclease-free water. The reactions were performed at 95  C for 1 min, followed by (95  C for 10 s, 59e63  C (annealing temperature of various studied genes) for 30 s, and melt curve 65e95  C with an increment of 0.05  C)  40 cycles. A negative template control (NTC) was added to eliminate any possibility of DNA contamination. The specificity of the PCR products was verified by melting curve analysis and gel electrophoresis. The primers were designed using the NCBI database and synthesized by Integrated DNA Technologies (Bangalore, India). The sets of forward and reverse primers of Tumor necrosis factor (TNF)-a, Interleukin (IL)-1b, IL-6, Nuclear factor kappa B (NF-kB), cyclooxygenase (COX)2 and GAPDH genes are given in Table 1. The raw fluorescence data were analyzed using Bio RAD Manager Software. The calculation was made using GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) as an endogenous control reference gene. The fold change in gene expression was calculated, taking into account the CT of the respective gene minus CT of an endogenous gene, followed by subtraction of the DCT value which gives DDCT. The fold change was then calculated by 2-DDCT.

2.10. DNA damage The level of DNA damage was assessed by alkaline single cell gel electrophoresis according to the technique given by Singh et al. with slight modification (Ahuja and Saran, 2001; Czarny et al., 2015; Singh et al., 1988). This technique allows the amount of single- and double-strand DNA breaks to be assessed, as well as the number of alkali labile sites. HOSE cells were obtained from culture after 7 days of OCPs exposure and without exposure. Total cell count and cell viability were evaluated using the Trypan blue exclusion method in a Neubauer chamber. The cell viability was found more than 90% in all OCPs exposed and control groups. 1  105 cells were diluted in agarose (1:2) and spread evenly on precoated 1% low melting agarose slides. On a third layer, 0.5% agarose was layered. The slides were treated with ice-cold cell lysis buffer (100 mM EDTA, 10 mM TRIS, 2.5 mM NaCl, 1% Triton X-100, pH 10, 10% DMSO) for 1 h in dark at 4  C. Then the slides were removed carefully and incubated into chilled electrophoresis buffer for 20 min. Further electrophoresis was carried out at 9 V for 30 min at 4  C in dark. And then the slides ware neutralized with 0.4 M Tris (PH 7.4). After neutralization, DNA was stained with ETBR and examined at 20e40 magnifications in a fluorescence microscope (B51, Olympus Japan) linked to the “Comet Assay Laboratory Universal Computer Image Analysis” software for image analysis. Negative and positive controls were used for each electrophoresis assay to ensure procedure reliability. The level of DNA Damage was measured based on the amount of DNA in the tail and the tail moment (Lu et al., 2017). Comet data has been analyzed by taking the average of Head DNA, tail DNA, tail moments and tail length.

Table 1 The sets of forward (F) and reverse (R) primers used in the real time PCR.

1

Gene Name

Accession number

Primer sequences (50 -30 )

Product length

TNF-a

NM_000594.3

201

IL-1b

NM_000576.2

IL-6

XM_005249745.5

NFkB

NM_001165412.2

COX-2

NM_000963.4

GAPDH

NM_001357943.2

F: GTG CTT GTT CCT CAG CCT CT R: GGG TTT GCT ACA ACA TGG GC F: TTG CTC AAG TGT CTG AAG CAG R: AGA TTC GTA GCT GGA TGC CG F: TTC AAT GAG GAG ACT TGC CTG G R: TTC CCC CAC ACC AAG TTG AG F: GCC ACC CGG CTT CAG AAT R: TAT GGG CCA TCT GTT GGC AG F: GAA AAC TGC TCA ACA CCG GAA R: AAG GGA GTC GGG CAA TCA TC F: CCA AGG TCA TCC ATG ACA ACT TTG GT R:TGT TGA AGT CAG AGG AGA CCA CCT G

Primers of each gene were designed using NCBI primer and synthesized by Integrated DNA Technologies (IDT, Bangalore, India).

180 195 144 293 381

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2.11. Statistical analysis

3.4. Effects of OCPs on pro-inflammatory cytokines expression

Each experiment was repeated independently three times in triplicates. Data shown are an average ± standard deviation. Statistical analyses were performed with SPSS v. 21. Comparisons of data obtained by one-way analysis of variance (ANOVA) followed by Bonferroni and Dunnet’s post hoc multiple comparison testing. IC50 analysis was carried out by GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). The level of probability are indicated as *P < 0.05 and **P < 0.01.

The relative gene expression of pro-inflammatory cytokines such as interleukin IL-6, IL-1b, and TNF-a, were studied. We have found significant high level of IL-6 mRNA expression in HOSE cells treated with b-HCH by 10.35 fold (p < 0.01), DDE by 10.82 fold (p < 0.01) and Dieldrin by 7.57 fold (p < 0.05) after 7 days as compared to DMSO control (Fig. 4A) and the amplified product was further run on the agarose gel electrophoresis as shown in the Fig. 4B which revealed OCPs exposure to HOSE cells increase the fold change of IL-6 gene compared to control. As shown in Fig. 4D, IL-1b mRNA expression significantly increased in HOSE cells treated with b-HCH by 2.75 fold (p < 0.01), DDE by 3.60 fold (p < 0.01) and Dieldrin by 2.38 fold (p < 0.05) after 7 days as compared to control (DMSO) and the agarose gel electrophoresis of real-time RT-PCR amplified product represented in Fig. 4E showed a high level of IL-1b expression in HOSE cells treated with b-HCH, DDE, and Dieldrin compared to control. In addition, TNF-a mRNA level found significantly high in HOSE cells following 7 days of b-HCH, DDE and Dieldrin exposure by 4.78 fold (p < 0.05), DDE by 4.31 fold (p < 0.05) and Dieldrin by 3.81 fold (p < 0.05) respectively as shown in Fig. 4F and their band intensity in agarose gel represent the high level of TNF-a mRNA expression in OCPs treated HOSE cells compared to control (DMSO) revealed in Fig. 4G.

3. Results 3.1. Effect of OCPs on cell viability and cytotoxicity HOSE cell viability was assessed by measuring MTT dye reduction assay after 3 and 7 days exposure to increasing concentration of b HCH, DDE, and Dieldrin. HOSE cells exposed to b HCH, DDE and Dieldrin revealed a decrease in cell viability with IC50 52.73, 70.04 and 63.77 mM respectively after 3 days and 48.77, 60.16 and 57.18 respectively after 7 days (Fig. 1A and B). HOSE cells exposure up to 20 mM concentration of b-HCH revealed >95% cell viability and exposure of DDE, Dieldrin up to 20 mM concentration revealed >100% cell viability following 3 and 7 days. Moreover, cell cytotoxicity was seen after 3 and 7 days following exposure of 30 mM concentration of b-HCH, DDE and Dieldrin and peaked after 100 mM (Fig. 1A and B). Hence, we selected 20 mM as the highest concentration for b HCH, DDE and Dieldrin in this study. These results suggest that exposure to b HCH, DDE and Dieldrin at high concentration (>30 mM) is toxic however, low concentration show approx. 100% cell viability of HOSE cells. 3.2. Impact of OCPs exposure on HOSE cell Impact of OCPs on the HOSE cell morphology was assessed after 7 days of exposure of 20 mM concentration of b-HCH, DDE and Dieldrin. Our results showed that in the control condition (DMSO) HOSE cells had cobblestone morphology (Fig. 2A) whereas HOSE cells exposed to b-HCH, DDE and Dieldrin exhibited poorly defined structures with increase in cellular granules (Fig. 2B, C & D). These observations show that an increased in granularity in HOSE cells may be due to inflammatory response stimulated by exposure to OCPs. 3.3. Intracellular ROS level in OCPs treated HOSE cells To determine whether OCPs treatments are associated with changes in intracellular ROS levels in HOSE cells, the relative level of intracellular ROS in HOSE cells was measured after treatment with 20 mM b-HCH, DDE and Dieldrin for 3 days using the redoxsensitive fluorescent dye 20 , 70 -dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma). H2DCFDA diffuses into the cells and is cleaved intracellularly by non-specific cellular esterases to nonfluorescent form 2, 7-dichlorodihydrofluorescein (DCFH), which is further rapidly oxidized by ROS and becomes a highly fluorescent compound 2, 7-dichlorofluorescein (DCF) (Myhre et al., 2003). The average fluorescent intensity of DCF is proportional to intracellular ROS levels. Thus obtained DCF fluorescent signals as an indicator of the ROS level are shown in Fig. 3 and the data are presented as percentage of control. All the OCPs such as b-HCH, DDE and Dieldrin significantly induced ROS level by 71.06%, 80.82% (p < 0.01) and 65.59% (p < 0.05) respectively compared to control (DMSO). In addition, positive control (50 mM H2O2) induced significantly ROS level by 78.44% (p < 0.01) over DMSO control.

3.5. Effect of OCPs on inflammatory cytokine modulators; transcription factor (NF-kB) and cyclooxygenase (COX-2) expression NF-kB gene expression was significantly induced in HOSE cells by b-HCH by 8.43 fold (p < 0.05), DDE by 11.66 (p < 0.01) and Dieldrin by 6.36 (p < 0.05) shown in Fig. 5A and their representative gel image are displayed in Fig. 5B that also showed a similar increase in band intensity as OCPs increase the fold change of NF-kB. Similarly, COX-2 mRNA expression was significantly upregulated in HOSE cells following 7 days exposure of b-HCH by 7.89 fold (p < 0.05), DDE by 11.52 fold (p < 0.05) and Dieldrin by 15.83 fold (p < 0.01) as demonstrated in Fig. 5C and their representative gel image revealed high band intensity of the COX-2 in HOSE cells treated with OCPs as compared to DMSO control as shown in Fig. 5D. 3.6. Effects of OCPs on IL-6 protein expression We have found that significant increase in the level of IL-6 protein expression in b-HCH (p < 0.01), DDE (p < 0.01) and Dieldrin (p < 0.01) treated HOSE cells as compared to control shown in Fig. 4C. Only IL-6 ELISA was performed due to its role in inflammatory response in the pathophysiology of ovarian cancer is more pronounced, however, other cytokines were not studied at protein level due to lack of funds. 3.7. Effect of OCPs in DNA damage Quantitative analysis for the alkaline comet assay showed significantly increased comet tail formation (tail length, tail intensity, and tail shape) after OCPs treatment in HOSE cells (Fig. 6) indicating DNA damage. The comet moment is the distance and the amount (measured in pixel density) of DNA that migrates from the center of the head of the comet. Fig. 7 shows the % of (A) Head DNA (B) tail DNA and (C) tail moment of the OCPs exposed and DMSO control groups. A significant difference was observed between OCPs treated HOSE cells (in reference to Head DNA, tail DNA and tail moment) and DMSO control (p < 0.01). The % head DNA significantly decrease in the cell treated with all three OCPs compared to

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Fig. 1. HOSE cell viability was assessed after 3 days (A) & 7 days (B) of OCPs exposure with increasing concentrations (from 1 mM to 200 mM) by MTT assay. MTT results are presented as a percentage of viability over DMSO treatment and each value is the mean ± S.D. of three separate experiments. IC50 of OCPs was calculated by GraphPad Prism7.

cells without OCPs (p < 0.01). Whereas, % tail DNA and the tail moment significantly increased in the cell treated with OCPs compared to DMSO control (p < 0.01). 4. Discussion In this study, we have investigated that organochlorine pesticide

(b HCH, DDE and Dieldrin) promote an inflammatory response in cultured human ovary surface epithelial (HOSE) cells. Dichlorodiphenyldichloroethylene (DDE), the metabolite of DDT is the most prevalent organochlorine residue present in human tissues (Jaga and Dharmani, 2003). We have found high level of ROS production and DNA damage along with up-regulation of proinflammatory cytokines such as TNF-a, IL-1b, IL-6, transcription

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Fig. 2. OCPs exposure-induced changes in HOSE cells. Cells were seeded in 12-well plates and after 50% confluence cells were exposed to (A) 0.25% DMSO (control) and 20 mM concentration of (B) b HCH (C) DDE, (D) Dieldrin for 3 days. Cells morphology was analyzed using an inverted microscope (life technology). Cellular granularity induced by OCPs represented in a white dotted rectangular box. Images selected are representative of three independent experiments. Photographs were performed with a magnification of  20.

Fig. 3. Effect OCPs on intracellular ROS production. HOSE cells (104 cells) were treated with b-HCH, DDE, and Dieldrin at a concentration of 20 mM for 3 days and in control HOSE cells treated with DMSO (0.25%). The percentage ROS level (OCPs/control*100) was determined and its levels in DMSO treated cells were set to 100 percent. Data are expressed as mean ± S.D. of triplicate determination from three independents experiments. Note: *p < 0.05 and **p < 0.01.

factor; NF-kB and enzyme; COX-2 in OCPs treated HOSE cells compared to control (DMSO). The best of our knowledge, this is the first report that explores the effect of b-HCH, DDE and Dieldrin in the inflammatory process and oxidative stress in human ovary epithelial cells by an in vitro study. Although, we found a high level of ROS generation, inflammatory cytokines expression, and DNA damage in HOSE cells following

7 days exposure of 1 mM and 10 mM concentration of b-HCH, DDE, and Dieldrin. However, DNA damage was not reached statistically significant level at both the concentration even 10 mM concentration of these OCPs showed significant high level of ROS and inflammatory cytokines in HOSE cells as compared to control. Also, DNA damage was not statistically significant level in HOSE cells following 3 days exposure to 20 mM concentration of these OCPs, though, ROS and inflammatory cytokines were significantly high (Figs. S1, S2, S3, and S4, Supplementary Material). While, 20 mM concentrations following 7 days exposure to these OCPs have shown a statistically significant effect on ROS generation, inflammatory cytokines overexpression as well as DNA damage with approx. 100% cell viability as compared to control (Figs. 3e5 and 7). Therefore, we focused to study long term effects of OCPs on ROS production, inflammatory process and DNA damage. Zucchini et al. studied various concentrations (0.2, 2, and 20 mM) of DDT, endosulfan, and heptachlor exposure to cultured primary human hepatocytes. They have found that 20 mM concentrations of these OCPs were non-cytotoxic as well as exhibit maximum effect on epithelial to mesenchymal transition, a process in liver carcinogenesis (Zucchini-Pascal et al., 2012). High level of pro-inflammatory cytokines & excessive production of ROS can damage DNA by overwhelming the antioxidant defense system. However, DNA damage is repaired by DNA repair mechanism (Barth et al., 2017). The efficacy of DNA repairs depends on the dose, duration as well as type of damaging agent and upon the species (Kaur and Kaur, 2018). Moreover, various human-made pollutants at environmental levels might lack an immediate effect, such as DNA damage (Hodgson et al., 2005). Hence, in the present study, lower dose and duration might not show a significant level of

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Fig. 4. Effect OCPs on pro-inflammatory cytokines. HOSE cells were treated with b HCH, DDE, and Dieldrin at 20 mM concentration for 7 days. (A) IL-6 (D) IL-1b (F) TNF-a mRNA levels were evaluated by real-time RT-PCR. The relative gene levels (normalized with respect to gapdh) were determined and IL-6, IL-1b & TNF-a level in DMSO treated cells set to 1. (C) IL-6 protein concentration was measured by ELISA. Data are expressed as mean ± S.D. of triplicate determination from three independents experiments. Note: *p < 0.05 and **p < 0.01. Real-time RT-PCR products of (B) IL-6, (E) IL-1b and (G) TNF-a gene were run on 1.6% agarose gel.

DNA damage. Nonetheless, upon the accumulation of ROS in HOSE cells following persistent exposure of OCPs might evade the repair system and increased the susceptibility to DNA damage that was revealed significantly high level at 20 mM following 7 days of exposure. Inflammation during ovulation produces a high level of inflammatory cytokines, ROS to facilitate growth, development, and remodeling of the follicle as well as repair of the ovulatory wound (Murdoch and Martinchick, 2004; Shan and Liu, 2009). However, during the lifetime, repetitive secretion of ROS, cytokines, and growth factors by the ovaries and immune cells make a chronic inflammatory micro-environment that initiate the malignant transformation of ovarian surface epithelial cells near to ovulation site in the ovary (Bahar-Shany et al., 2014; Tone et al., 2012). Moreover, earlier study showed that inflammation due to increase in production of ROS, cytokines and oxidative stress in the ovary may increase risk of epithelial ovarian cancer in women having pelvic inflammatory disease (PID), endometriosis, Polycystic Ovarian Syndrome (PCOS), obesity or frequent exposure to talc and

asbestos (Savant et al., 2018). Today environmental pollution is a huge problem and therefore is a global concern. There are various pathological conditions including cancer arise due to environmental pollutants since they disturb the body physiological process. Environmental pollutants such as (i) vinclozolin, (ii) NP, (iii) phthalates and (iv) atrazine are associated with carcinogenesis by dysregulation of NFkB, IL-6, COX2, and TNF-a, respectively (Thompson et al., 2015). As shown in Fig. 3, b HCH, DDE and Dieldrin exposure significantly increased ROS generation in HOSE cells. ROS associated with a wide variety of cancers including ovary cancer (Chan et al., 2008). Mostly, pesticide molecules induce DNA damage via the production of ROS (Bagchi et al., 1995). In addition, ROS can exclusively activate signaling pathways that contribute to tumor development (Storz, 2005) such as oxidative stress switch on pro-survival signal in the cell by dysregulating RAS signaling result in activation of NF-kB and upregulation of its downstream targets such as IL-1b, IL-6, and IL-8 (Lane et al., 2011). ROS exposure could potentially lead to transformative changes of epithelial cells in the ovary, as demonstrated

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Fig. 5. Effect OCPs on cytokine modulators. HOSE cells were treated with b HCH, DDE, and Dieldrin at 20 mM concentration for 7 days. (A) NFkB (C) COX-2 mRNA levels were measured by real-time RT-PCR. The relative gene levels (normalized with respect to gapdh) were determined and NFkB & COX-2 levels in DMSO treated cells were set to 1. Data are expressed as mean ± S.D. of triplicate determination from three independents experiments. Note: *p < 0.05 and **p < 0.01. (B) & (D) represent the real-time RT-PCR product of NFkB and COX-2 gene run on 1.6% agarose gel.

Fig. 6. Representative images of HOSE cells after the treatment of OCPs. The alkaline comet assay was used to compare OCPS induced DNA double-strand breaks in cells. HOSE cells treated with 0.25% DMSO (A) and 20 mM of b HCH (B), DDE (C) and Dieldrin (D) after 7 days alkaline comet assay was performed and the representative image was taken at magnification x20.

for ovarian surface epithelium cells grown in 3D culture (King et al., 2013). In vitro study suggests that Inflammation caused by DDT increases ROS (Jin et al., 2014) and COX-2 (Han et al., 2008) which are hallmarks of cancer. Besides, we found significantly overexpression of inflammatory cytokines such as TNF-a, IL-1b, and IL-6 in the HOSE cells following 7 days of OCPs exposure as shown in Fig. 4. Ovary epithelial cells secrete inflammatory cytokines such as TNF-a, IL-1b, and IL-6 that promote stromal cells and/or immune cells to secrets more inflammatory cytokines which mold inflammation in the local tumor microenvironment (Kulbe et al., 2012; Maccio and Madeddu, 2012). This signifies the sustainable exposure of OCPs to HOSE cells may

cause secretion of pro-inflammatory cytokines expression in the ovary that may create tumor microenvironment. IL-1 induces gene expression of TNF-a which act as mitogen for ovarian surface epithelium (Wu et al., 1992). The up-regulation of the inflammatory cytokine IL-1, TNF-a, and predominantly IL-6 play a central role in the progression of EOC (Kulbe et al., 2012). Many studies showed IL6 signaling plays a crucial role in tumor initiation, promotion, and progression (Lane et al., 2011). IL-6 is one of the major immunoregulatory cytokines present in the EOC microenvironment and is involved in the autocrine growth of ovarian cancer cells (Rabinovich et al., 2007). IL-6 has been associated with poor survival and emerging as a potential therapeutic target for EOC. Tumor

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Fig. 7. Effects of OCPs on DNA damage in HOSE cells. HOSE cells were treated with b HCH, DDE, and Dieldrin at 20 mM concentration for 7 days. (A) % Head DNA, (B) % tail DNA and (C) % tail moment were evaluated by alkaline comet assay and analyzed more than 100 cells per treatment. Data are expressed as median with a range of triplicate determination from three independents experiments. Note: **p < 0.01.

necrosis factor (TNF-a), is the key mediator of inflammation and promotes tumor initiation and progression (Yan et al., 2006). Ovarian cancer cells secrete high levels of TNF-a compared to normal ovarian epithelial cells resulting in autocrine up-regulation of TNF-a mRNA and IL6 (Kulbe et al., 2007; Szlosarek et al., 2006). Furthermore, the present study revealed significant upregulation of inflammatory mediators such as NF-kB and COX-2 gene expression in the HOSE cells exposed to b HCH, DDE, and Dieldrin (Fig. 5). TNF-a is a strong inducer of COX-2 expression by stimulating the NF-kB system (Seo et al., 2004). TNF-a binding to its receptor activates the NF-kB pathway which results in the upregulation of inflammatory cytokines (IL-1b, IL-6), COX-2, and their sustainable production trigger uncontrolled NF-kB expression (Candido and Hagemann, 2013; Colotta et al., 2009; Feldmann and Maini, 2008). The continuous activity of NF-kB could promote the production of ROS, pro-inflammatory cytokines, and COX-2 thereby damage DNA of surrounding epithelial cells leading to the tumor microenvironment and promote tumor progression (Grivennikov et al., 2010). NF-kB is associated with epithelial ovarian cancer with co-expression of IL-6 (Hoesel and Schmid, 2013).

Cyclooxygenase (COX)-2 is an early response gene in inflammation and play an important role during carcinogenesis (Seo et al., 2004; Vendramini-Costa and Carvalho, 2012). The COX-2 enzymes were the first identified molecular targets in tumor-associated inflammation. COX-2 is overexpressed in acute & chronic inflammation as well as in tumors (Hanahan and Weinberg, 2011). COX-2 overexpression has been shown to induce genetic instability (Singh et al., 2007) as well as reported in several human cancers including ovary cancer (Gu et al., 2008; Wu and Sun, 2015; Yokouchi and Kanazawa, 2015). We also observed the high level of DNA damage in HOSE cells exposed to b-HCH, DDE and Dieldrin. Genetic instability is one of the crucial mechanisms of cancer initiation due to DNA damage (Hanahan and Weinberg, 2011). DDT promotes DNA damage & genetic instability (Yanez et al., 2004). Besides, DDT causes micronucleus formation, sister chromatid exchange (Gomez-Arroyo et al., 2000). It has been revealed that the accumulation of genetic damage in epithelial ovary cells by hormonal surges and inflammation with each ovulation cycle eventually leads to the development of ovarian cancer (Fathalla, 1971). Environmental pollutants

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such as pesticides and polycyclic aromatic hydrocarbons (PAH) induce DNA damage and play a crucial role in carcinogenesis through direct interaction, ROS and inflammatory process (Barth et al., 2017; Irigaray and Belpomme, 2010). If DNA repair mechanisms are inefficient, that results in the accumulation of lesions in ~ oz and Albores, 2011). DNA and might induce carcinogenesis (Mun This is in the supports of the present findings that revealed the sustained exposure of OCPs residues in the HOSE cells induce ROS production, inflammatory cytokines and its mediator that can lead to DNA damage may, in turn, enhance the risk of malignant transformation. It has been revealed that increased IL-6, TNF-a, levels along with oxidative stress and DNA damage in women with PCOS (Dinger et al., 2005; Tarkun et al., 2006). PCOS is a common endocrine disorder that is associated with epithelial ovarian cancer (Schildkraut et al., 1996). b-HCH (Beard and Rawlings, 1999), DDT (Bulayeva and Watson, 2004), Dieldrin (Soto et al., 1994) act as endocrine-disrupting chemicals interfering with the endocrine function. The risk of epithelial ovarian cancer may increase with increased oxidative stress and DNA damage observed in women with PCOS. Also, OSE derived cytokines and growth factors including IL-1 & IL-6 act on the inclusion cysts and induce changes in gene expression that facilitate neoplasia (Ziltener et al., 1993). Inclusion cysts are less plasticity than normal and are more prone to ovarian carcinogenesis. Ovarian pathologies such as PCOS and endometriosis are more frequent among women having pesticide exposure (Zhang et al., 2014). Hence, from the present study, it assist that neoplastic progression of HOSE cells present in the inclusion cyst may be promoted by tumor-promoting microenvironment arise due to persistent exposure to OCP residues in the ovary. In this study, we have also measured cytokine IL-6 at protein and mRNA expression levels using ELISA and RT-qPCR respectively. However, IL-1b and TNF-a were measured only at mRNA expression levels. The primary reason was the lack of funds. However, this did not affect our result and interpretation. The high specificity, sensitivity, and stability of RT-qPCR assay are alone efficient to show the simultaneous measurement of mRNA expression (Nagaeva et al., 2002; Winberg et al., 2016). A major strength of this investigation is that the human ovary surface epithelial (HOSE) cells were procured and all the experiment was conducted at passage 2 and 3 where cells maintained their epithelial properties as suggested by literature (Science Cell, Germany). Another strength is the real-time RT-qPCR analyses of mRNA for all chosen cytokines were done at the same time to avoid experimental bias. 5. Conclusion Consequently, we conclude that persistent exposures of b-HCH, DDE and Dieldrin to human ovary surface epithelial cells induce ROS generation, and secrete various pro-inflammatory cytokines this lead to chronic inflammatory reaction and form tumorpromoting microenvironment further potentiate genetic instability (DNA damage). All these results at least account for the carcinogenic potential of b-HCH, DDE and Dieldrin in the transformation of epithelial ovary cells. Author contributions H.K.S, T.S. and B.D.B designed the research. H.K.S performed the experiments and analyzed the data. T.S assisted in cell procurement and primer design. H.K.S wrote the manuscript. T.S & B.D.B gave valuable comments that improved the manuscript. All authors contributed to the final version of the manuscript.

Declaration of competing interest The authors of this manuscript have nothing to declare. Acknowledgments This research was funded by Department of Biotechnology (DBT), Govt. of India. Grant ID:BT/PR7913/MED/30/964/2013. One of the author, HKS is thankful to Indian Council of Medical Research (ICMR), Govt. of India for providing the individual SRF fellowship support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125691. References Ahuja, Y.R., Saran, R., 2001. Potential of single cell gel electrophoresis assay (comet assay) in heavy ion radiation biology. Int. J. Hum. Genet. 1 (2), 151e156. https:// doi.org/10.1080/09723757.2001.11885751. Alavanja, M.C., Bonner, M.R., 2012. Occupational pesticide exposures and cancer risk: a review. J. Toxicol. Environ. Health B Crit. Rev. 15 (4), 238e263. https:// doi.org/10.1080/10937404.2012.632358. Bagchi, D., Bagchi, M., Hassoun, E.A., Stohs, S.J., 1995. In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 104 (1e3), 129e140. Bahar-Shany, K., Brand, H., Sapoznik, S., Jacob-Hirsch, J., Yung, Y., Korach, J., Levanon, K., 2014. Exposure of fallopian tube epithelium to follicular fluid mimics carcinogenic changes in precursor lesions of serous papillary carcinoma. Gynecol. Oncol. 132 (2), 322e327. https://doi.org/10.1016/j.ygyno.2013.12.015. Barth, A., Brucker, N., Moro, A.M., Nascimento, S., Goethel, G., Souto, C., …, Garcia, S.C., 2017. Association between inflammation processes, DNA damage, and exposure to environmental pollutants. Environ. Sci. Pollut. Res. Int. 24 (1), 353e362. https://doi.org/10.1007/s11356-016-7772-0. Beard, A.P., Rawlings, N.C., 1999. Thyroid function and effects on reproduction in ewes exposed to the organochlorine pesticides lindane or pentachlorophenol (PCP) from conception. J. Toxicol. Environ. Health 58 (8), 509e530. Bulayeva, N.N., Watson, C.S., 2004. Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways. Environ. Health Perspect. 112 (15), 1481e1487. https://doi.org/10.1289/ehp.7175. Candido, J., Hagemann, T., 2013. Cancer-related inflammation. J. Clin. Immunol. 33 (Suppl. 1), S79eS84. https://doi.org/10.1007/s10875-012-9847-0. Chan, D.W., Liu, V.W., Tsao, G.S., Yao, K.M., Furukawa, T., Chan, K.K., Ngan, H.Y., 2008. Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells. Carcinogenesis 29 (9), 1742e1750. https:// doi.org/10.1093/carcin/bgn167. Colotta, F., Allavena, P., Sica, A., Garlanda, C., Mantovani, A., 2009. Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 30 (7), 1073e1081. https://doi.org/10.1093/carcin/bgp127. Czarny, P., Kwiatkowski, D., Kacperska, D., Kawczynska, D., Talarowska, M., Orzechowska, A., Sliwinski, T., 2015. Elevated level of DNA damage and impaired repair of oxidative DNA damage in patients with recurrent depressive disorder. Med. Sci. Monit. 21, 412e418. https://doi.org/10.12659/MSM.892317. Dinger, Y., Akcay, T., Erdem, T., Ilker Saygili, E., Gundogdu, S., 2005. DNA damage, DNA susceptibility to oxidation and glutathione level in women with polycystic ovary syndrome. Scand. J. Clin. Lab. Investig. 65 (8), 721e728. Fathalla, M.F., 1971. Incessant ovulation–a factor in ovarian neoplasia? Lancet 2 (7716), 163. https://doi.org/10.1016/s0140-6736(71)92335-x. Federico, A., Morgillo, F., Tuccillo, C., Ciardiello, F., Loguercio, C., 2007. Chronic inflammation and oxidative stress in human carcinogenesis. Int. J. Cancer 121 (11), 2381e2386. https://doi.org/10.1002/ijc.23192. Feldmann, M., Maini, S.R., 2008. Role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeutics. Immunol. Rev. 223, 7e19. https:// doi.org/10.1111/j.1600-065X.2008.00626.x. Fleming, J.S., Beaugie, C.R., Haviv, I., Chenevix-Trench, G., Tan, O.L., 2006. Incessant ovulation, inflammation and epithelial ovarian carcinogenesis: revisiting old hypotheses. Mol. Cell. Endocrinol. 247 (1e2), 4e21. https://doi.org/10.1016/ j.mce.2005.09.014. Gomez-Arroyo, S., Diaz-Sanchez, Y., Meneses-Perez, M.A., Villalobos-Pietrini, R., De Leon-Rodriguez, J., 2000. Cytogenetic biomonitoring in a Mexican floriculture worker group exposed to pesticides. Mutat. Res. 466 (1), 117e124. https:// doi.org/10.1016/s1383-5718(99)00231-4. Grivennikov, S.I., Karin, M., 2011. Inflammatory cytokines in cancer: tumour necrosis factor and interleukin 6 take the stage. Ann. Rheum. Dis. 70 (Suppl. 1), 104e108. https://doi.org/10.1136/ard.2010.140145. Grivennikov, S.I., Greten, F.R., Karin, M., 2010. Immunity, inflammation, and cancer. Cell 140 (6), 883e899. https://doi.org/10.1016/j.cell.2010.01.025.

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