Arch Toxicol DOI 10.1007/s00204-014-1359-7

MOLECULAR TOXICOLOGY

Endosulfan induces COX‑2 expression via NADPH oxidase and the ROS, MAPK, and Akt pathways Hyung Gyun Kim · Young Ran Kim · Jin Hee Park · Tilak Khanal · Jae Ho Choi · Minh Truong Do · Sun Woo Jin · Eun Hee Han · Young Ho Chung · Hye Gwang Jeong 

Received: 21 May 2014 / Accepted: 28 August 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Endosulfan (1,4,5,6,7,7-hexachloro-8,9,10-trinorborn -5-en-2,3-ylenebismet-hylene) is correlated with endocrine disruption, reproductive, and immune dysfunctions. Recently, endosulfan was shown to have an effect on inflammatory pathways, but its influence on cyclooxygenase-2(COX-2) expression is unclear. This study investigated the effects of COX-2 and molecular mechanisms by endosulfan in murine macrophage RAW 264.7 cells. Endosulfan significantly induced COX-2 protein and mRNA levels, as well as COX-2 promoter-driven luciferase activity and the production of prostaglandin E2, a major COX-2 metabolite. Transfection experiments with several human COX-2 promoter constructs revealed that endosulfan activated NF-κB, C/EBP, AP-1, and CREB. Moreover, Akt and mitogen-activated protein kinases (MAPK) were significantly activated by endosulfan. Moreover, endosulfan increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2, and NOX3. Endosulfan-induced Akt/MAPK pathways and

Hyung Gyun Kim and Young Ran Kim have contributed equally to this article. Electronic supplementary material  The online version of this article (doi:10.1007/s00204-014-1359-7) contains supplementary material, which is available to authorized users. H. G. Kim · Y. R. Kim · J. H. Park · T. Khanal · J. H. Choi · M. T. Do · S. W. Jin · H. G. Jeong (*)  Department of Toxicology, College of Pharmacy, Chungnam National University, 220 Gung‑dong, Yuseong‑Gu, Daejeon 305‑764, Republic of Korea e-mail: [email protected] E. H. Han · Y. H. Chung  Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea

COX-2 expression were attenuated by DPI, a specific NOX inhibitor, and the ROS scavenger N-acetylcysteine. These results demonstrate that endosulfan induces COX-2 expression via NADPH oxidase, ROS, and Akt/MAPK pathways. These findings provide further insight into the signal transduction pathways involved in the inflammatory effects of endosulfan. Keywords  Endosulfan · COX-2 · ROS · NADPH oxidase

Introduction In recent years, chemicals, such as pesticides, have been introduced into the environment and it attracted particular attention due to exposure of the general population. Endosulfan (1,4,5,6,7,7-hexachloro-8,9,10-trinorborn-5-en-2,3ylenebismethylene) sulphite, a mixture of stereoisomers α and β at a ratio of 2:1, has been used widely as a broad-spectrum cyclodiene insecticide (Omurtag et al. 2008). Endosulfan consists of two diastereomers, α and β; α-endosulfan exists as two asymmetrical, twist-chair enantiomers which interchange, while β-endosulfan has a symmetrical-chair conformation (Schmidt et al. 2014). Endosulfan is absorbed through ingestion, inhalation, and skin contact. It is toxic to fish and aquatic invertebrates (Naqvi and Vaishnavi 1993), and has been implicated in mammalian gonadal toxicity (Sinha et al. 1997), genotoxicity (Chaudhuri et al. 1999), and neurotoxicity (Silva and Gammon 2009). Recently, we reported that endosulfan upregulates the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in macrophages, mediated in part through NF-κB (Han et al. 2007). The production of NO and inflammatory cytokines is of considerable interest in studies aimed at understanding the toxicity

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of endosulfan on wildlife and human health. The effects of endosulfan on inflammation are unclear despite its widespread use as a pesticide and evidence showing that it can modulate immune function. Also, the influence of endosulfan on expression of cyclooxygenase-2 (COX-2) and the inflammatory responses of macrophages remains unclear. COX catalyses the synthesis of prostaglandins (PGs) from arachidonic acid. The two COX isozymes are encoded by different genes, COX-1 and COX-2. The COX-1 isozyme is a housekeeping protein that is present in most tissues and catalyses the synthesis of PGs for normal physiological functions (Kam and See 2000). In contrast, COX-2 is not present under normal physiological conditions, but is rapidly induced by various tumour promoters, growth factors, cytokines, mitogens, and carcinogens in various cell types (Prescott and Fitzpatrick 2000). Some studies have indicated that the expression of COX-2 is mediated by the activation of the mitogen-activated protein kinases (MAPK), Akt (Hsieh et al. 2006). In addition, reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and oxygen free radicals have been implicated in initiating inflammatory responses in macrophages through the activation of transcription factors, such as NF-κB and AP-1, and other signal transduction pathways, such as MAPK, leading to enhanced gene expression of proinflammatory mediators (Turpaev 2002; Fialkow et al. 2007). The NOX family of NADPH oxidases includes seven different enzymes whose main function is the production of ROS. These enzymes are expressed widely in numerous tissues and play various roles, including cell signalling, gene expression regulation, cell death, differentiation, and growth (Brown and Griendling 2009). In addition, recent literature suggests that NADPH oxidases may be the source of ROS-mediated COX-2 up-regulation (Sancho et al. 2011). Here, we hypothesised that endosulfan induces COX-2 expression via NADPH oxidase/ROS and Akt/MAPK pathways. These findings provide further insight into the signal transduction pathways involved in the inflammatory effects of endosulfan.

Materials and methods Reagents and antibodies Chemicals and cell culture materials were obtained from the following sources: endosulfan was a mixture of alphaand beta-isomers at a ratio of 2:1, lucigenin, NADPH, and phorbol 12-myristate 13-acetate (PMA) from Sigma Chemical Co. (St. Louis, MO, USA); anti-COX-2 antibody and enzyme immunoassay reagents for the PGE2 assays from Cayman Chemical Co. (Ann Arbour, MI, USA); SYBR® Safe DNA Gel Stain kit and Dulbecco’s Modified Eagle’s

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Arch Toxicol

Medium (DMEM), foetal bovine serum (FBS), and penicillin–streptomycin solution from Life Technologies, Inc. (Carlsbad, CA, USA); WST-1 assay kit from Roche (Roche, London, UK); luciferase assay system from Promega (Madison, WI, USA); pCMV-β-gal from Clontech (Palo Alto, CA, USA); LipofectAMINE 2000 from Invitrogen Co. (Carlsbad, CA, USA); antibodies to β-actin, Nucleus p65, Lamin B, IκB-α, and phospho-p65 (Ser 536) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); protein assay kit from Bio-Rad Laboratories, Inc. (Hercules, CA, USA); primary antibodies (anti-phospho-MAP kinase (Erkl/2; Thr202/Tyr204), anti-phospho-p38 MAP kinase (Thr180/Tyr182), anti-phospho-SAPK/JNK MAP kinase (Thr183/Tyr185), anti-phospho-Akt (Ser473), phosphoIκB-α (Ser 32), and secondary antibody (horseradish peroxidase [HRP]-linked anti-rabbit and anti-mouse IgG) from Cell Signaling Technology (Danvers, MA, USA); ECL chemiluminescence system and polyvinylidene difluoride (PVDF) membranes from Amersham Pharmacia Biotech (Uppsala, Sweden); PD98059, SB20358, SP600125, and LY294002 from Calbiochem (La Jolla, CA, USA). Polymerase chain reaction (PCR) oligonucleotide primers were custom-synthesised by Bioneer Co. (Korea). All chemicals were of the highest grade commercially available. Cell culture and treatment RAW 264.7 cells, a mouse macrophage cell line, were obtained from the American Type Culture Collection (Bethesda, MD, USA) and grown in DMEM supplemented with 10 % FBS, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5 % CO2 humidified incubator. The endosulfan was dissolved in dimethyl sulfoxide (DMSO), and stock solutions were added directly to the culture media. Control cells were treated only with solvent. The final concentration of solvent never exceeded 0.1 % and did not affect the assay systems. Cell viability assay Cell cytotoxicity was examined using a WST-1 assay and LDH leakage assay kit (Roche, London, UK), according to the manufacturer’s instructions. Briefly, 5 × 105 RAW 264.7 cells/well in 10 % FBS/DMEM were seeded into 96-well plates. Endosulfan (0.5–100 µM) was added to the wells, and the plates were incubated at 37 °C. The presence of endosulfan did not interfere at this wavelength. PGE2 production RAW 264.7 cells were incubated with endosulfan or 100 nM PMA. After incubating the cells for 24 h, the culture medium was collected, and PGE2 in culture medium

Arch Toxicol

was measured using a specific enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions. PGE2 concentration was determined by measuring absorbance at 415 nm. ROS production ROS production in RAW 264.7 cells was measured using the redox-sensitive fluorescent dye DCFDA. After treatment with endosulfan or vehicle, cells were incubated with 25 μM DCFDA for 20 min. The cells were rinsed twice with DMEM containing 1 % FBS, and fluorescence was detected on a FL600 fluorescence spectrophotometer (GeminiXS, Molecular Devices) with excitation and emission set at 490 and 530 nm, respectively. Determination of NADPH oxidase activity Cells were grown in six-well culture plates and incubated with endosulfan for the indicated time intervals. Cells were gently scraped and centrifuged for 10 min at 4 °C. The cell pellet was resuspended in 35 mL of ice-cold DMEM medium per vial and then kept on ice. Five microlitres of the cell suspension was added to a final volume of 200 mL of pre-warmed DMEM medium containing either NADPH (1 μM) or lucigenin (20 μM), to initiate the reaction, followed by immediate measurement of chemiluminescence using a luminometer (Appliskan, Thermo). Quantitative real‑time PCR Cells were cultured with either endosulfan or 100 nM PMA for 16 h. Total RNA was extracted from treated cells using RNAiso Reagent (TaKaRa Bio, Otsu, Japan), according to the manufacturer’s protocol. cDNA synthesis and semi-quantitative real-time PCR for COX-2, NOX 1–4 and β-actin mRNA were performed, and the PCR product formation was continuously monitored during the PCR using Sequence Detection System software, version 1.7 (Applied Biosystems, Foster City, CA, USA). The expression level of COX-2 in the exposed cells was compared to the expression level in control cells at each collection time point using the comparative cycle threshold (Ct) method by monitoring the increase of the reporter dye (SYBR) (Applied Biosystems, 1997). The quantity of each transcript was calculated according to the instrument manual, and normalised to the amount of β-actin, a housekeeping gene. Western blotting RAW 264.7 cells were cultured with either endosulfan or 100 nM PMA for 30 min (for Akt), 60 min (IκB-α degradation), 30 min (for MAPK), 3 h (nuclear p65 translocation),

6 h (c-jun, c-fos, CREB and C/EBP β) or 16 h (COX-2). Equal amounts of total cellular protein (50 μg) were resolved by 10 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After blocking, the membranes were incubated with the target antibody. An HRP-conjugated secondary antibody to IgG was used. Immunoreactive proteins were visualised using the ECL Western blot detection system. Transfection and β‑galactosidase assays The human (h)COX-2 promoter-driven luciferase deletion (−1432/+59, −327/+59, −220/+59, and −124/+59) and mutant (mNF-κB, mC/EBP, and mAP-1/CRE) constructs were generous gifts from Dr. Hiroyasu Inoue (Nara Women’s University, Nara, Japan) and have been described elsewhere (Wadleigh et al. 2000). mNF-κB is the −327/+59 COX-2 promoter construct with a mutant NF-κB binding site. mC/EBP is the −327/+59 COX-2 promoter construct with a mutant C/EBP binding site. mAP-1/CRE is the −327/+59 COX-2 promoter construct with mutant AP-1 and CRE binding sites. pNF-κB-Luc, pC/EBP-Luc, pAP-1-Luc, and pCRE-Luc were purchased from Stratagene (La Jolla, CA, USA). In each well, 1 µg each of test plasmid and pCMVβ-gal plasmid DNA was introduced into the cells using 0.5-μL LipofectAMINE 2000, according to the manufacturer’s instructions. For each well, 1-µg plasmid DNA was introduced into the cells using 1-μL LipofectAMINE 2000, according to the manufacturer’s instructions. After 4 h, the medium was replaced with basal medium. The cells were then treated with either endosulfan or PMA for 24 h and lysed. The luciferase activity was normalised to the β-galactosidase activity and expressed relative to the activity of control cells. Statistical analysis All data are presented as the means of the three independent experiments, each performed in triplicate. A one-way analysis of variance (ANOVA) was used to determine the significance of differences between treatment groups. The Newman–Keuls test was used for multi-group comparisons. Statistical significance was assigned for p values