Novel Lipid-Soluble Thiol-Redox Antioxidant and Heavy Metal Chelator, N,N&#39;-bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aortic Endothelial Cells

Secor, J. D.; Kotha, S. R.; Gurney, T. O.; Patel, R. B.; Kefauver, N. R.; Gupta, N.; Morris, A. J.; Haley, B. E.; Parinandi, N. L.

International Journal of Toxicology http://ijt.sagepub.com/ Novel Lipid-Soluble Thiol-Redox Antioxidant and Heavy Metal Chelator, N,N′ -bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aortic Endothelial Cells Jordan D. Secor, Sainath R. Kotha, Travis O. Gurney, Rishi B. Patel, Nicholas R. Kefauver, Niladri Gupta, Andrew J. Morris, Boyd E. Haley and Narasimham L. Parinandi International Journal of Toxicology 2011 30: 619 originally published online 12 October 2011 DOI: 10.1177/1091581811422413 The online version of this article can be found at: http://ijt.sagepub.com/content/30/6/619 Published by: http://www.sagepublications.com On behalf of: American College of Toxicology Additional services and information for International Journal of Toxicology can be found at: Email Alerts: http://ijt.sagepub.com/cgi/alerts Subscriptions: http://ijt.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav >> Version of Record - Jan 6, 2012 OnlineFirst Version of Record - Oct 12, 2011 What is This? Downloaded from ijt.sagepub.com by guest on September 17, 2012 International Journal of Toxicology 30(6) 619-638 Novel Lipid-Soluble Thiol-Redox ª The Author(s) 2011 Reprints and permission: sagepub.com/journalsPermissions.nav Antioxidant and Heavy Metal Chelator, DOI: 10.1177/1091581811422413 http://ijt.sagepub.com N,N0-bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aortic Endothelial Cells Jordan D. Secor1, Sainath R. Kotha1, Travis O. Gurney1, Rishi B. Patel1, Nicholas R. Kefauver1, Niladri Gupta2, Andrew J. Morris3, Boyd E. Haley2, and Narasimham L. Parinandi1 Abstract Here, we investigated thiol-redox-mediated phospholipase D (PLD) signaling as a mechanism of mercury cytotoxicity in mouse aortic endothelial cell (MAEC) in vitro model utilizing the novel lipid-soluble thiol-redox antioxidant and heavy metal chelator, N,N0 -bis(2-mercaptoethyl)isophthalamide (NBMI) and the novel PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). Our results demonstrated (i) mercury in the form of mercury(II) chloride, methylmercury, and thimerosal induced PLD activation in a dose- and time-dependent manner; (ii) NBMI and FIPI completely attenuated mercury- and oxidant-induced PLD activation; (iii) mercury induced upstream phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) leading to downstream threonine phosphorylation of PLD1 which was attenuated by NBMI; (iv) mercury caused loss of intracellular glutathione which was restored by NBMI; and (v) NBMI and FIPI attenuated mercury- and oxidant-induced cytotoxicity in MAECs. For the first time, this study demonstrated that redox-dependent and PLD-mediated bioactive lipid signaling was involved in mercury-induced vascular EC cytotoxicity which was protected by NBMI and FIPI. Keywords mercury, vasculotoxicity, PLD, endothelial cell, NBMI, thiol redox, antioxidant, FIPI, mercaptoethylisophthalamide, bioactive lipid signaling Introduction Mercury (Hg), a transition element, is a serious environmental 1 Lipid Signaling, Lipidomics, and Vasculotoxicity Laboratory, Division of heavy metal pollutant with established toxicity in humans. Pulmonary, Allergy, Critical Care and Sleep Medicine, Dorothy M. Davis Heart Mercury has been reported to cause cytotoxicity, neurotoxicity, and Lung Research Institute and Division of Pharmacology, Colleges of Med- and immunotoxicity with no known nutritional actions and icine and Pharmacy, The Ohio State University, Columbus, OH, USA 2 CTI Science, Inc., Lexington, KY, USA physiological role.1–3 Mercury enters the air, water, and soil 3 Medicine and Molecular and Cellular Biochemistry, University of Kentucky, environments from the industrial processing, medical uses, and Lexington, KY, USA anthropogenic activities. Inorganic Hg undergoes biomethyla- tion leading to the formation of methylmercury which is a Corresponding Author: highly toxic form of the element.4 Methylmercury bioaccumu- Narasimham L. Parinandi, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Dorothy M. Davis Heart and Lung Research Institute, Room # lates in the aquatic food chain which results in Hg toxicity in 611-A, 473 West 12th Avenue, College of Medicine, The Ohio State Univer- fish and mammalian systems. Methylmercury has been shown sity, Columbus, OH 43210, USA to cause hypertension in rats, and mental retardation and Email: narasimham.parinandi@osumc.edu Downloaded from ijt.sagepub.com by guest on September 17, 2012 620 International Journal of Toxicology 30(6) cerebral palsy in infants.5 Methylmercury is known to cause can be hypothesized that the hydrophobic cell membrane oxidative stress in biological systems through the generation of phospholipid domain is a prime target for the adverse actions reactive oxidant species (ROS).6,7 Human exposure to the sig- of Hg. This is further supported by our earlier report that Hg nificantly more toxic methylmercury through environmental induces membrane phospholipid hydrolysis through the activa- contamination is well documented.8 Mercury vapor released tion of PLD and PLA2.7,22,31 Mercury is also known to alter/ from the elemental Hg (Hg0) in dental amalgams is the chief deplete the thiol-redox status, induce oxidative stress, and com- source of Hg exposure among dental patients.9 In the human plex with protein ligands in biological systems.32 In order to mouth, elemental Hg is biotransformed into methylmercury, an counteract this at cellular level, a multifunctional pharmaco- organic toxic form of the metal. Thimerosal, an ethylmercury logical compound with antioxidant properties, thiol-redox compound used as a preservative in influenza and other vac- protection, and heavy metal chelating ability are needed. cines, poses controversies and concerns as an elicitor of autism Unlike the commonly used water-soluble thiol protectants and and neurological defects in infants and children.10–14 In addi- heavy metal chelators such as N-acetyl-L-cysteine (NAC), tion to dental amalgams and herbal medicines, cosmetics 2,3-dimercaptopropane-1-sulfonate (DMPS), and meso-2,3- (beauty products) have been recently reported to cause nonoc- dimercaptosuccinic acid (DMSA), 33 a hydrophobic and cupational exposure of humans to Hg.15 lipid-soluble heavy metal chelator would be ideal to sequester Exposure to Hg is emerging as a risk factor associated with Hg in the phospholipid membrane bilayer microenvironment the cardiovascular diseases (CVDs) in humans.16–20 Levels of of the cell (Figure 1A, B). A lipid-soluble hydrophobic com- Hg in toenail and urine have been directly correlated with the pound, N,N0 -bis(2-mercaptoethyl)isophthalamide (NBMI), elevated risk of myocardial infarction and coronary heart has been synthesized for use in remediation of Hg-polluted disease. 5,18,19 Mercury exposure has also been linked to soil and water environs (Figure 1C).34 N,N0 -bis(2-Mercap- asthma and hypertension.21 Regardless of the reported asso- toethyl)isophthalamide appears to possess unique features of ciation of exposure to Hg with CVD in humans, the role of lipid solubility and lodging in the membrane phospholipid vascular endothelial cells (ECs) in the Hg-mediated CVD is bilayer, Hg chelation, and thiol-redox stabilization at the cell not known. Vascular endothelium is crucial for the structure membrane level. and function of the blood vessels toward maintaining the Therefore, here, we utilized NBMI to dissect out the homeostasis of the circulatory system.22 The dysfunction of thiol-redox-dependent activation of PLD signaling and its EC has been shown to cause vascular damage and leak and mediation of Hg cytotoxicity in vascular ECs. The currently the breakdown of the cardiovascular system. 23,24 Hence, available novel PLD-specific inhibitor, 5-fluoro-2-indolyl Hg-induced vasculotoxicity at the EC level appears to be des-chlorohalopemide (FIPI), allowed us to establish the role involved in the Hg-induced CVDs. of PLD and PLD-derived bioactive lipid mediators in the Phospholipase D (PLD) is a crucial signaling enzyme ubi- Hg-induced lipid signaling and cytotoxicity in vascular ECs quitous in mammalian cells, including vascular ECs, which in the present study (Figure 1D). Water-soluble thiol antiox- generates the bioactive lipid signal mediators, phosphatidic idants and heavy metal chelators (NAC and DMSA) at higher acid (PA), diacylglycerol (DAG), and lysophosphatidic acid concentrations are required for in vivo treatment of heavy (LPA) from the hydrolysis of membrane phospholipids.25–27 metal toxicity as they are cleared from the body in urine more These bioactive lipids regulate important cellular functions readily as their lipid-soluble counterparts. In addition, small- such as cell proliferation and differentiation.25,26 Earlier, we molecule thiol protectants, such as NAC, are known to act as have reported that Hg induces the activation of PLD in bovine pro-oxidants and exacerbate the oxidative stress. However, pulmonary artery ECs (BPAECs) which is associated with the lipid-soluble antioxidants such as NBMI and the lipid- loss of thiols, ROS generation, and intracellular calcium.7,22 soluble PLD inhibitor (FIPI) are more likely to accumulate Earlier, we have also demonstrated that oxidant-induced PLD in lipid microdomains of the cells (membranes) and tissues signaling is regulated by extracellular-regulated kinase 1/2 (fat deposits) and can be mobilized to the sites of action. (ERK1/2) in vascular ECs. 25 However, the thiol-redox- Cumulatively, it is anticipated that the benefit of using the mediated upstream PLD-signaling mechanism and downstream lipid-soluble thiol protectant such as NBMI and the PLD- Hg-induced cytotoxicity in vascular ECs are not known. There- specific inhibitor, FIPI, for in vivo treatment of heavy fore, here we hypothesized that Hg would induce PLD activa- metal-mediated oxidative stress and toxicity, would offer the tion through thiol-redox-regulated upstream ERK1/2 requirement of lower doses of those compounds in addition to activation, which would further lead to the formation of the their longer duration of action relative to water-soluble thiol bioactive lipid signal mediator, PA, mediating the cytotoxicity antioxidants and heavy metal chelators (NAC and DMSA). of Hg in vascular ECs. For the first time, our current study demonstrated that the Hg- Plasma membranes of animal cells rich in phospholipids induced PLD activation in mouse aortic ECs (MAECs) was are the prime targets for toxicants such as Hg. Both the inor- thiol-redox dependent and ERK regulated. Also, the current ganic and organic forms of Hg have been shown to bind to study revealed that both NBMI and FIPI offered protection the phospholipids, in addition to the –SH-containing proteins against the Hg-induced cytotoxicity, establishing the role of in the animal cell membranes28–30 and thus cause alterations thiol redox and PLD-generated bioactive lipid signal mediator in the structure and function of cell membrane. Therefore, it (PA) therein. Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 621 A B NAC DMSA C NBMI D FIPI Figure 1. Chemical structure of pharmacological compounds. A, N-Acetyl-L-cysteine (NAC). B, meso-2,3-Dimercaptosuccinic acid (DMSA). C, N,N0 -bis(2-Mercaptoethyl)isopthalamide (NBMI). D, 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI). Materials and Methods (GSH), mercury(II) chloride, methylmercury chloride, thimerosal, NAC, dimethylsulfoxide (DMSO), DMSA, and Materials analytical reagents of highest purity were purchased from The MAECs used in this study were provided by Dr Robert Sigma Chemical Co (St Louis, Missouri). 4-Hydroxy-2- Auerbach at the University of Wisconsin, Madison, Wisconsin. nonenal (4-HNE) was obtained from Biomol International Phosphate-buffered saline (PBS) was obtained from Biofluids L.P. (Plymouth Meeting, Pennsylvania). PD98059 were Inc (Rockville, Maryland). Minimal essential medium (MEM), obtained from Calbiochem (San Diego, California). Primary nonessential amino acids, trypsin, fetal bovine serum (FBS), antibodies for PLD1, PLD2, phosphotheonine-PLD1, ERK-1, penicillin/streptomycin, phosphate-free modified Dulbecco- ERK-2, and phospho-ERK-1/2 raised in rabbit were obtained modified Eagle medium (DMEM), DMEM high glucose, tis- from Cell Signaling Technology, Inc (Danvers, Massachu- sue culture reagents, 3-[4,5-dimethylthiazol-2-yl]-2,5- setts). Phosphatidylbutanol (PBt) was obtained from Avanti diphenyl tetrazolium bromide reduction kit (MTT assay kit), Polar Lipids (Alabaster, Alabama). [32P]Orthophosphate (car- lactate dehydrogenase (LDH) cytotoxicity assay kit (LDH rier-free) was obtained from New England Nuclear (Wilming- release assay kit), t-octylphenoxypolyethoxyethanol (Triton ton, Delaware). Anti-rabbit AlexaFluor 488-conjugated X-100), bovine serum albumin (BSA), diamide, glutathione antibody and 40 ,6-diamidino-2-phenylindole dihydrochloride Downloaded from ijt.sagepub.com by guest on September 17, 2012 622 International Journal of Toxicology 30(6) (DAPI) were purchased from Molecular Probes Invitrogen Cell Culture Co (Carlsbad, California). Glutathione chemiluminescence The MAECs were cultured in DMEM (high glucose) supple- assay kit (GSH-Glo) was obtained from Promega Corporation mented with 5% (volume/volume [vol/vol]) FBS, 100 U/mL (Madison, Wisconsin). Paraformaldehyde was purchased penicillin and streptomycin, and 1% (vol/vol) nonessential from Electron Microscopy Sciences (Fort Washington, amino acids at 37" C under a humidified 95% air–5% carbon Pennsylvania). Polyoxyethylene sorbitan monolaurate dioxide (CO2) atmosphere, according to our previously pub- (Tween-20) was purchased from Bio-Rad Laboratories lished method.40 Cells in culture were maintained at 37" C in a (Hercules, California). Horseradish peroxidase (HRP)-conju- humidified environment of 95% air–5% CO2 and grown to gated anti-rabbit secondary antibody and the enhanced che- contact-inhibited monolayers with typical cobblestone mor- miluminescence (ECL) kit for the detection of proteins by phology. When confluence was reached, cells were trypsinized Western blotting were obtained from Amersham (Arlington and subcultured in T 75-cm2 flasks or 12.5 or 35 or 100 mm Heights, Illinois). Diperoxovanadate (DPV) was a generous sterile tissue culture dishes or 96-well sterile tissue culture gift from Dr Ramasarma of the Indian Institute of Sciences, plates. Confluent cells showed cobblestone morphology under Bangalore, India. light microscope. All experiments were conducted between 8 and 20 passages (80%-90% confluence). All media and treat- ments were carefully adjusted to pH 7.4 before use. N,N0 -bis(2-Mercaptoethyl)Isophthalamide Synthesis N,N0 -bis(2-Mercaptoethyl)isophthalamide was synthesized Assay of PLD Enzyme Activity using a modification of the method of Matlock et al.34,35 Three grams of 2-aminoethylthiol hydrochloride was dissolved in Phospholipase D activity was quantified using our established 25 mL of chloroform and 3.7 mL of triethylamine and placed method of measuring the formation of [ 32P]-radiolabeled in an ice bath with stirring; 2.68 g of isophthaloyl chloride was PBt.27,39 Cellular lipids were extracted and PBt was isolated dissolved in 25 mL of chloroform and slowly added to the using our published methods of lipid extraction and thin layer solution containing 2-aminoethylthiol and allowed to stir for chromatographic separation, respectively. Radioactivity was 2 hours on ice. Precipitation of the NBMI was induced by measured using liquid scintillation counting and quantified as adding about 100 mL of 0.1 N hydrochloric acid (HCl) slowly DPM normalized to 106 counts in the total cellular lipid extract to the stirring mixture. The resulting precipitate was collected or as percentage of control (vehicle-treated cells). by filtration and washed 2 times with a water–chloroform (50/50) mixture and then 2 times with 0.1 N HCl and 3 times Lactate Dehydrogenase Release Assay for Cytotoxicity with distilled water. The resulting white powder was dried under vacuum and the yielded the product NBMI, showing a Cytotoxicity in MAECs was determined by assaying the extent 70% yield. Gram amounts of this powder were dissolved in of release of LDH from cells according to our previously pub- pure ethanol and recrystallized twice resulting in the final prod- lished method.39 At the end of treatment, LDH released into the uct. The purity of NBMI was determined by liquid chromato- medium was determined spectrophotometrically using the graphy–mass spectrometry (LC-MS)/MS. Column was a commercial LDH assay kit according to the manufacturer’s Waters X-Bridge C-18 (150 mm ! 3.0 mm, 5 mm particle size; recommendations (Sigma Chemical Co). Waters Corporation, Milford, MA). The mobile phase con- sisted of (A) aqueous with 0.1% formic acid and (B) methanol MTT Reduction Assay for Cytotoxicity with 0.1% formic acid. A gradient system was used and the Cytotoxicity in MAECs was determined by assaying the extent total run time was 30 minutes. The elution conditions expressed of reduction in MTT in intact cells using the commercial MTT as percentage of B is as follows: 0 to 30 seconds, 10% B; 30 reduction assay kit as previously reported.24 At the end of the seconds to 10 minutes, 10% to 90% B; 10 to 19 minutes, 90% experimental treatments, MTT solution (10% vol/vol in MEM) B; 19 to 20 minutes, 90% to 10% B; and 20 to 30 minutes, 10% was added and the cells were incubated for 3 hours, following B. Injection volume was 10 mL. Flow rate was 250 mL/min and which MTT solvent was added in an amount equal to the orig- the retention time for NBMI was 9.81 minutes. Analysis was inal culture volume. Absorbance of the reduced MTT was done on a Varian LC 1200 L Triple Quadruple MS using a determined spectrophotometrically, according to the manufac- positive electrospray ionization (ESI) source. Chemicals used turer’s recommendations (Sigma Chemical Co). in the LC-MS/MS analysis were purchased from Sigma- Aldrich, St Louis, Missouri. N,N 0 -bis(2-Mercaptoethyl)i- sophthalamide used in the current study was of >99.8% purity. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blotting Preparation of cell lysates and Western blotting were carried 5-Fluoro-2-Indolyl des-Chlorohalopemide Synthesis out according to our previously published methods.25 Protein 5-Fluoro-2-indolyl des-chlorohalopemide was prepared as content in the cell lysates was determined by the BCA protein described earlier.36–39 assay (Thermo Scientific, Rockford, IL). Cell lysates Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 623 containing 40 mg of protein were loaded onto a 10% sodium DMSO concentration in the cell treatment medium was 0.1% dodecyl sulfate–polyacrylamide gel electrophoresis (SDS- (vol/vol). All other solutions of water-soluble pharmacological PAGE) gel and proteins were separated at 90 V for 2.5 hours. compounds were freshly prepared in MEM for treatment of cells. Proteins resolved on gels were electrotransferred onto the poly- vinylidene difluoride (PVDF) membranes at 200 mA for 2 Statistical Analysis hours at 4" C. The PVDF membranes with proteins were then washed with 0.05% Tween-20 in Tris-buffered saline (TBST) All experiments were done in triplicate. Data were expressed as following which they were blocked with TBST containing 5% mean + standard deviation (SD). Statistical analysis was carried milk for 1 hour and then incubated with the primary antibody out by analysis of variance (ANOVA) using SigmaStat (Version (1:1000 dilution) at 4" C overnight in TBST containing 5% 2.0, Jandel). The level of statistical significance was taken as P < .05. BSA. Membranes were then treated with HRP-conjugated anti-rabbit immunoglobulin G ([IgG] 1:2000 dilution) in TBST containing 5% milk at room temperature for 1 hour followed by Results washing for 3 times with TBST. The immunoblots were then N,N0 -bis(2-Mercaptoethyl)Isophthalamide Attenuates developed with the ECL reagents, according to the manufac- turer’s recommendations. Images on films were scanned and Hg-Induced PLD Activation in MAECs quantified using ImageJ software. Earlier, we have reported that Hg in 3 different forms (inor- ganic mercury(II) chloride, organic methylmercury, and phar- maceutical thimerosal) induces PLD activation in BPAECs.7 Confocal Immunofluorescence Microscopy However, in the current study, we used the MAEC model of Translocation of PLD1 and PLD2 were analyzed by confocal aortic origin which is different from the earlier used BPAEC laser scanning microscopy according to our earlier published model of arterial origin. Therefore, here, we investigated method.24 The MAECs grown on sterile glass coverslips (75% whether Hg in 3 different forms (mercury(II) chloride, methyl- confluence) were subjected to desired treatments and then were mercury, and thimerosal) would cause activation of PLD in rinsed 3 times with PBS containing 0.01% Tween-20 (PBST) and MAECs similar to that observed in BPAECs as reported by fixed with 3.4% paraformaldehyde in PBS for 5 minutes at room us earlier.7 In the current study, we further investigated whether temperature. The cells were then rinsed 3 times with PBST and the novel thiol-redox antioxidant and heavy metal chelator, permeabilized with 0.25% Triton-X-100 prepared in PBST for 5 NBMI, would attenuate the Hg-induced PLD activation in minutes. The cells were then washed 3 times with PBST 0.01% MAECs. One hour following treatment of cells, mercury(II) and treated with 1% BSA blocking buffer for 30 minutes at room chloride, methylmercury, and thimerosal induced significant temperature. The cells were incubated overnight at room tem- activation of PLD ([32P]PBt formation) in a dose-dependent perature with the primary antibody (anti-PLD1 and anti-PLD2 manner in comparison to untreated control cells. Mercury(II) antibodies at 1:200 dilution) in 1% BSA solution in PBST. After chloride induced 2-, 4-, and 10-fold, methylmercury induced rinsing 3 times with PBST, the cells were treated with AlexaFluor 0.5-, 2-, and 3-fold, and thimerosal induced 5-, 10-, and 12-fold 488-conjugated antibody (1:100 dilution) in 1% BSA in PBST increase in PLD activation in a dose-dependent fashion at 5, 10, for 1 hour. The cells were washed 3 times with PBST 0.01%, and 25 mmol/L dose, respectively, as compared to untreated mounted, and examined under Zeiss LSM 510 Confocal/Multi- control cells (Figure 2A). N,N0 -bis(2-Mercaptoethyl)isophtha- photon Microscope at 490-nm excitation and 530-nm emission lamide (10, 25, and 50 mmol/L) significantly attenuated PLD under !63 magnification. The images were captured digitally. activation induced by mercury(II) chloride (25 mmol/L), methylmercury (10 mmol/L), and thimerosal (25 mmol/L) in MAECs at 1 hour of exposure (Figure 2B-D). N,N0 -bis(2-Mer- Glutathione Determination captoethyl)isophthalamide was effective in attenuating the Intracellular soluble thiol (GSH) levels were determined using mercury(II) chloride-induced PLD activation (48%, 65%, and the GSH-Glo assay kit as reported earlier.39 The MAECs 86% inhibition at 10, 25, and 50 mmol/L, respectively) as com- grown up to 90% confluence in 96-well sterile plates were pared to cells treated with mercury(II) chloride alone. The treated with MEM alone or MEM containing desired concen- methylmercury-induced [32P]PBt formation was also attenu- trations of treatments under a humidified 95% air–5% CO2 ated by NBMI (67%, 53%, and 72% inhibition at 10, 25, and atmosphere. Following incubation, intracellular GSH levels 50 mmol/L, respectively) as compared to cells treated with were determined according to the manufacturer’s recommen- methylmercury alone. N,N0 -bis(2-Mercaptoethyl)isophthala- dations (Promega Corp, Madison, Wisconsin). mide also attenuated the thimerosal-induced PLD activation (58%, 76%, and 80% inhibition at 10, 25, and 50 mmol/L, respectively) as compared to cells treated with thimerosal Preparation of Solutions Containing Pharmacological Agents alone. These results showed that organic, inorganic, and phar- Stock solutions of the hydrophobic pharmacological agents maceutical Hg significantly induced PLD activation in MAECs including NBMI, FIPI, and PD98059 were freshly prepared in in a dose-dependent manner. Also, the results revealed that the DMSO and diluted in MEM for treatment of cells. The final novel thiol-redox antioxidant and heavy metal chelator, NBMI, Downloaded from ijt.sagepub.com by guest on September 17, 2012 624 International Journal of Toxicology 30(6) A B 30000 Control 14000 Vehicle 5 µM [32P] PBt Formed (DPM/Dish) * NBMI (10 µM) [32P] PBt Formed (DPM/Dish) 25000 * 10 µM 12000 NBMI (25 µM) 25 µM NBMI (50 µM) * 10000 20000 * * 8000 15000 ** 6000 ** 10000 * 4000 * * 5000 * * 2000 ** 0 0 HgCl2 MeHg Thimerosal Control HgCl2 (25 µM) C D 16000 Vehicle NBMI (10 µM) [32P] PBt Formed (DPM/Dish) 14000 * NBMI (25 µM) 8000 Vehicle [32P] PBt Formed (DPM/Dish) NBMI (50 µM) NBMI (10 µM) 12000 7000 * NBMI (25 µM) 10000 6000 NBMI (50 µM) 5000 8000 4000 6000 ** ** 3000 ** 4000 ** 2000 ** ** 2000 1000 0 0 Control MeHg (10 µM) Control Thimerosal (25 µM) Figure 2. N,N0 -bis(2-Mercaptoethyl)isopthalamide (NBMI) attenuates the mercury-induced phospholipase D (PLD) activation in a dose- dependent fashion in mouse aortic endothelial cells (MAECs). The MAECs (5 ! 105 cells/35 mm dish) were labeled with [32P]orthophosphate in phosphate-free Dulbecco-modified Eagle medium (DMEM) for 12 hours. Following [32P]orthophosphate labeling, MAECs were treated with minimal essential medium (MEM) alone or MEM containing different concentrations (5, 10, and 25 mmol/L) of mercury(II) chloride, methylmer- cury, and thimerosal for 1 hour (A) in the presence of 0.05% (volume/volume [vol/vol]) 1-butanol. The MAECs were also labeled with [32P]orthophosphate, then pretreated with MEM or different concentrations of MEM containing NBMI (10, 25, and 50 mmol/L) for 1 hour, then treated with mercury(II) chloride (25 mmol/L) (B); methylmercury (10 mmol/L) (C); and thimerosal (25 mmol/L); (D), for 1 hour in the presence of 0.05% (vol/vol) 1-butanol. At the end of the incubation period, [32P] PBt formed was determined. Data represent mean + standard deviation (SD) calculated from 3 independent experiments. *Significantly different at P < .05 as compared to cells treated with MEM alone. **Significantly different at P < .05 as compared to cells treated with MEM containing mercury alone. significantly attenuated the Hg-induced PLD activation in a comparable to equal concentrations of NAC treatment (66%- dose-dependent manner in MAECs. 69% of the methylmercury-induced activation and 73%-81% of the thimerosal-induced activation at 50 mmol/L, respectively) N,N0 -bis(2-Mercaptoethyl)Isophthalamide Attenuates in comparison to MAECs treated with methylmercury (10 mmol/L) and thimerosal (25 mmol/L) alone for 1 hour Hg-Induced PLD Activation With Efficacy Similar to (Figure 3A, B). The NBMI showed marginally greater attenua- the Established Thiol Protectants tion of Hg-induced PLD activation than equal concentrations of Earlier, we reported that the Hg-induced PLD activation in DMSA (74%-72% of the methylmercury-induced activation BPAECs is thiol-redox dependent.7 We have also shown that and 66%-43% of the thimerosal-induced activation at the widely used thiol protectants NAC and DMSA attenuate the 50 mmol/L, respectively) as compared to MAECs treated with Hg-induced PLD activation in BPAECs.7 Therefore, here, we methylmercury (10 mmol/L) and thimerosal (25 mmol/L) alone investigated the efficacy of NBMI, the novel thiol-redox anti- for 1 hour (Figure 3C, D). These results demonstrated that the oxidant, by comparison with 2 well-established thiol novel thiol-redox antioxidant, NBMI, was equally effective in protectants, NAC and DMSA, in attenuating the Hg-induced attenuating the Hg-induced PLD activation in MAECs as the 2 activation of PLD in MAECs. The NBMI treatment offered well-established thiol protectants, NAC and DMSA, tested here significant attenuation of the Hg-induced PLD activation under identical conditions. Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 625 A B 7000 Vehicle * NBMI (50 µM) 18000 Vehicle 6000 * [32P] PBt Formed (DPM/Dish) NAC (50 µM) NAC (5 mM) NBMI (50 µM) [32P] PBt Formed (DPM/Dish) 16000 NAC (50 µM) 5000 14000 NAC (5 mM) 4000 12000 10000 3000 ** ** 8000 2000 6000 ** ** 4000 ** 1000 2000 ** 0 0 Control MeHg (10 µM) Control Thimerosal (25 µM) C D 9000 Vehicle 6000 Vehicle * 8000 NBMI (50 µM) * NBMI (50 µM) [32P] PBt Formed (DPM/Dish) [32P] PBt Formed (DPM/Dish) DMSA (50 µM) 5000 DMSA (50 µM) 7000 DMSA (5 mM) DMSA (5 mM) 6000 4000 5000 ** 3000 4000 ** 2000 ** 3000 ** ** 2000 ** 1000 1000 0 0 Control MeHg (10 µM) Control Thimerosal (25 µM) Figure 3. N,N 0 -bis(2-Mercaptoethyl)isopthalamide (NBMI) attenuates the mercury-induced phospholipase D (PLD) activation in similar fashion to N-acetyl- L -cysteine (NAC) and meso-2,3-dimercaptosuccinic acid (DMSA) in mouse aortic endothelial cells (MAECs). The MAECs (5 ! 105 cells/35 mm dish) were labeled with [32P]orthophosphate in phosphate-free Dulbecco-modified Eagle medium (DMEM) for 12 hours. Following [32P]orthophosphate labeling, cells were pretreated with minimal essential medium (MEM) alone or MEM containing NBMI (50 mmol/L) and MEM containing NAC (50 mmol/L, A and 5 mmol/L, B) or DMSA (50 (C) mmol/L and 5 mmol/L (D)) for 1 hour. After pretreatment, cells were treated with MEM alone or MEM containing methylmercury (10 mmol/L) or thimerosal (25 mmol/L) for 1 hour in the presence of 0.05% (volume/volume [vol/vol]) 1-butanol. At the end of the incubation period, [32P]phosphatidylbutanol ([32P]PBt) formed was determined. Data represent mean + standard deviation (SD) calculated from 3 independent experiments. *Significantly different at P < .05 as compared to cells treated with MEM alone. **Significantly different at P < .05 as compared to cells treated with MEM containing mercury alone. N,N0 -bis(2-Mercaptoethyl)Isophthalamide Attenuates L) for 1 hour caused significant (7- and 5-fold, respectively) Oxidant-Induced PLD Activation activation of PLD as compared to untreated control cells (Figure 4A, B). The NBMI treatment showed significant Oxidants have been shown to activate EC PLD through thiol- dose-dependent attenuation of PLD activation induced by redox-mediated regulation.26 Thiol protectants have been 4-HNE (39%, 68%, and 74% inhibition at 10, 25, and shown to attenuate the oxidant-induced PLD activation in 50 mmol/L, respectively) and DPV (59%, 77%, and 75% inhi- ECs.26 Earlier, we reported that the Hg-induced PLD activation bition at 10, 25, and 50 mmol/L, respectively) in MAECs as in BPAECs is mediated by ROS and oxidative stress.7 Conse- compared to cells treated with the same oxidants alone. These quently, here, we investigated whether the novel thiol-redox results demonstrated that oxidants such as 4-HNE and DPV antioxidant, NBMI, would attenuate the oxidant-induced induced PLD activation and the novel thiol-redox antioxidant, PLD activation in ECs. Treatment of MAECs with the well- NBMI, effectively attenuated the oxidant-induced PLD activa- established oxidants 4-HNE (100 mmol/L) and DPV (10 mmol/ tion in MAECs in a dose-dependent fashion. Downloaded from ijt.sagepub.com by guest on September 17, 2012 626 International Journal of Toxicology 30(6) activation in MAECs as determined by [32P]PBt formation. A 5-Fluoro-2-indolyl des-chlorohalopemide (250, 500 nmol/L, 5000 Vehicle and 1 mmol/L) exhibited significant, dose-dependent attenua- 4500 * NBMI (10 µM) tion of the mercury(II) chloride-induced PLD activation (77%, NBMI (25 µM) [32P] PBt Formed (DPM/Dish) 4000 NBMI (50 µM) 80%, and 88% at 250, 500 nmol/L, and 1 mmol/L, respectively) 3500 as compared to MAECs treated with mercury(II) chloride 3000 (25 mmol/L) alone for 1 hour (Figure 5A). Methylmercury- ** induced PLD activation was also attenuated by FIPI (43%, 2500 52%, and 59% at 250 nmol/L, 500 nmol/L, and 1 mmol/L, 2000 ** ** respectively) as compared to MAECs treated with methylmer- 1500 cury (10 mmol/L) alone for 1 hour (Figure 5B). Furthermore, 1000 FIPI attenuated the thimerosal-induced PLD activation (62%, 500 59%, and 65% at 250, 500 nmol/L, and 1 mmol/L, respectively) 0 as compared to cells treated with thimerosal (25 mmol/L) alone Control 4-HNE (100 µM) for 1 hour (Figure 5C). These results revealed that the novel PLD-specific inhibitor, FIPI, effectively attenuated the Hg- B induced PLD activation in MAECs in a dose-dependent 5000 Vehicle manner. 4500 * NBMI (10 µM) [32P] PBt Formed (DPM/Dish) 4000 NBMI (25 µM) NBMI (50 µM) Phospholipase D-Specific Inhibitor, FIPI, Attenuates 3500 3000 Oxidant-Induced PLD Activation 2500 It has been shown that oxidants and Hg induce PLD activation 2000 ** and FIPI, the PLD-specific inhibitor, attenuates the 1500 bleomycin-induced PLD activation in vascular ECs.26,39 Our ** ** 1000 earlier experiments in the current study also revealed that FIPI 500 significantly attenuated the Hg-induced PLD activation in 0 MAECs. Accordingly, here, we conducted studies to establish Control DPV (10 µM) the specificity of FIPI to inhibit the oxidant- and agonist 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD acti- Figure 4. N,N0 -bis(2-Mercaptoethyl)isopthalamide (NBMI) attenuates vation in MAECs. Following pretreatment with FIPI for 12 the oxidant-induced phospholipase D (PLD) activation in mouse aortic hours, MAECs were treated with oxidants (hydrogen peroxide endothelial cells (MAECs). The MAECs (5 ! 105 cells/35 mm dish) [H 2 O 2 ], 100 mmol/L, diamide, 100 mmol/L, or 4-HNE, were labeled with [32P]orthophosphate in phosphate-free Dulbecco- 100 mmol/L) for 1 hour. TPA was used as an agonist to induce modified Eagle medium (DMEM) for 12 hours. Following [32P]ortho- phosphate labeling, cells were pretreated with minimal essential PLD activation through protein kinase C (PKC)–signaling medium (MEM) alone or MEM containing different concentrations pathway.41 TPA-induced PLD activation was attenuated by (10, 25, and 50 mmol/L) NBMI for 1 hour. Following NBMI pretreat- FIPI in a dose-dependent manner (89%, 91%, and 87% by ment, cells were treated with 4-hydroxy-2-nonenal ([4-HNE] 250, 500 nmol/L, and 1 mmol/L FIPI, respectively) as com- 100 mmol/L; A) or diperoxovanadate ([DPV] 10 mmol/L; B) for 1 hour pared to cells treated with TPA alone (Figure 6A). The FIPI, in the presence of 0.05% (volume/volume [vol/vol]) 1-butanol. At the at the same doses, significantly attenuated PLD activation end of the incubation period, [32P]phosphatidylbutanol ([32P]PBt) induced by H 2 O 2 (53% and 45% attenuation at 250 and formed was determined. Data represent mean + standard deviation (SD) calculated from 3 independent experiments. *Significantly differ- 500 nmol/L, respectively), diamide (48%, 56%, and 44% ent at P < .05 as compared to cells treated with MEM alone. **Signif- attenuation at 250, 500 nmol/L and 1 mmol/L, respectively), icantly different at P < .05 as compared to cells treated with MEM and 4-HNE (37%, 42%, and 50% attenuation at 250, containing oxidant alone. 500 nmol/L, and 1 mmol/L, respectively) as compared to cells treated with the same oxidants alone for 1 hour (Figure 6B-D). Phospholipase D-Specific Inhibitor, FIPI, Attenuates These results revealed that the novel PLD-specific inhibitor, FIPI, attenuated the agonist- and oxidant-induced PLD acti- Hg-Induced PLD Activation vation in a dose-dependent manner in MAECs. Earlier, we have demonstrated that the novel PLD-specific inhibitor, FIPI, attenuates the bleomycin-induced PLD activa- N,N0 -bis(2-Mercaptoethyl)Isophthalamide Attenuates tion in lung microvascular ECs.39 However, the effectiveness of FIPI in attenuating the Hg-induced PLD activation in ECs Hg-Induced PLD1 and PLD2 In Situ Translocation has not been reported so far. Therefore, here, we conducted Our earlier study revealed that pro-oxidants like vitamin C studies to establish the efficacy of the newly available PLD- induce translocation of PLD1 and PLD2 isoforms in lung specific inhibitor, FIPI, in attenuating the Hg-induced PLD microvascular ECs.25 Also, we have reported that Hg induces Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 627 PLD activation in BPAECs through ROS formation, oxidative A stress, and thiol alteration.7 Hence, here, we investigated whether Hg would induce in situ translocation of PLD1 and 12000 Vehicle PLD2 isoforms and whether NBMI would attenuate the process * FIPI (250 nM) in MAECs. In untreated control MAECs, little to no PLD [32P] PBt Formed (DPM/Dish) FIPI (500 nM) 10000 FIPI (1 µM) staining was visible as revealed by confocal immunofluores- 8000 cence microscopy imaging. However, both methylmercury and thimerosal caused enhanced translocation of both PLD1 6000 and PLD2 isoforms in situ in MAECS. Also, NBMI treatment 4000 showed significant attenuation of the Hg-induced PLD1 and ** ** PLD2 translocation in cells. These results demonstrated that 2000 ** Hg caused PLD translocation in situ in MAECs which was effectively attenuated by the novel thiol-redox antioxidant, 0 Control HgCl2 (25 µM) NBMI (Figure 7). B 5000 Vehicle Mercury Induces Phosphorylation of ERK1/2 and PLD1 4500 * FIPI (250 nM) Oxidants and metals have been shown to induce phosphoryla- [32P] PBt Formed (DPM/Dish) FIPI (500 nM) 4000 FIPI (1 µM) tion and activation of ERK1/2 in different cellular systems.42,43 3500 Also, it has been reported that oxidants activate PLD in ECs 3000 ** through phosphorylation of PLD1 mediated by upstream acti- 2500 ** vation and phosphorylation of ERK1/2.25 Therefore, here, we 2000 ** investigated whether Hg would induce phosphorylation of 1500 ERK1/2 which in turn would mediate the phosphorylation of 1000 PLD1 in MAECs. The MAECs were treated with Hg (methyl- 500 mercury, 10 mmol/L or thimerosal, 25 mmol/L) for 0 to 60 0 minutes and then examined for phosphorylation of ERK1/2 Control MeHg (10 µM) and threonine phosphorylation of PLD1 by SDS-PAGE and Western blotting. Methylmercury induced a 1.56-fold C increase in ERK1/2 phosphorylation at 5 minutes and 1.44- 4000 * Vehicle and 1.23-fold increase at 15 and 30 minutes, respectively FIPI (250 nM) (Figure 8A). Methylmercury also induced 2.17-, 2.71-, and [32P] PBt Formed (DPM/Dish) 3500 FIPI (500 nM) 3000 FIPI (1 µM) 5.02-fold PLD1 threonine phosphorylation at 5, 15, and 30 minutes of methylmercury treatment, respectively, in MAECs 2500 relative to untreated control cells (Figure 8B). Thimerosal 2000 ** induced 1.21- and 1.59-fold increase in ERK1/2 phosphoryla- 1500 ** ** tion at 5 and 60 minutes, respectively, relative to untreated 1000 control cells (Figure 8C). Also, thimerosal induced 4.80- and 500 10.00-fold increase in PLD1 phosphorylation at 5 and 60 min- 0 utes of treatment, respectively, relative to untreated control Control Thimerosal (25 µM) cells (Figure 8D). These results demonstrated that mercury induced robust phosphorylation of ERK1/2 at the initial time Figure 5. Phospholipase D (PLD)-specific inhibitor, 5-fluoro-2-indolyl of treatment, whereas the induction of intense threonine phos- des-chlorohalopemide (FIPI), attenuates the mercury-induced PLD acti- phorylation of PLD1 occurred at later times of treatment, vation in mouse aortic endothelial cells (MAECs). The MAECs suggesting activation of ERK1/2 upstream of PLD1 threonine (5 ! 105 cells/35 mm dish) were labeled with [32P]orthophosphate in phosphorylation. phosphate-free Dulbecco-modified Eagle medium (DMEM) alone or phosphate-free DMEM containing different concentrations (250, 500 nmol/L, and 1 mmol/L) of FIPI for 12 hours. Following [32P]orthopho- Extracellular-Regulated Kinase 1/2 Upstream Inhibitor, sphate labeling, cells were treated with minimal essential medium (MEM) alone or MEM containing mercury(II) chloride (25 mmol/L; A), methyl- PD98059, Attenuates Hg-Induced PLD Activation mercury (10 mmol/L; B) and thimerosal (25 mmol/L; C) for 1 hour in the Several studies have shown ERK1/2 to act as an upstream presence of 0.05% (volume/volume [vol/vol]) 1-butanol. At the end of activator of PLD in intracellular signaling pathways. 25,42 the incubation period, [32P]phosphatidylbutanol ([32P]PBt) formed was determined. Data represent mean + standard deviation (SD) calculated Therefore, here, we investigated the modulatory effect of from 3 independent experiments. *Significantly different at P < .05 as ERK1/2-signaling inhibition on the Hg-induced PLD activa- compared to cells treated with MEM alone. **Significantly different at tion using the ERK1/2 upstream inhibitor, PD98059, and mea- P < .05 as compared to cells treated with MEM containing mercury alone. suring (i) [32P]PBt formation, (ii) ERK1/2 phosphorylation, Downloaded from ijt.sagepub.com by guest on September 17, 2012 628 International Journal of Toxicology 30(6) A B 8000 Vehicle * FIPI (250 nM) 2500 Vehicle 7000 [32P] PBt Formed (DPM/Dish) FIPI (500 nM) * [32P] PBt Formed (DPM/Dish) FIPI (250 nM) 6000 FIPI (1 µM) 2000 FIPI (500 nM) 5000 1500 4000 ** 3000 1000 ** 2000 500 1000 ** ** ** 0 0 Control H2O2 (100 µM) Control TPA (25 nM) C D 900 Vehicle 1200 Vehicle * FIPI (250 nM) FIPI (250 nM) 800 * [32P] PBt Formed (DPM/Dish) FIPI (500 nM) [32P] PBt Formed (DPM/Dish) 1000 FIPI (500 nM) 700 FIPI (1 µM) FIPI (1 µM) 600 800 500 ** ** ** ** ** 600 400 † ** 300 400 † 200 † 200 100 0 0 Control Diamide (100 µM) Control 4-HNE (100 µM) Figure 6. 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI) attenuates the oxidant-induced phospholipase D (PLD) activation in mouse aortic endothelial cells (MAECs). The MAECs (5 ! 105 cells/35 mm dish) were labeled with [32P]orthophosphate in phosphate-free Dulbecco-modified Eagle medium (DMEM) alone or phosphate-free DMEM containing different concentrations (250, 500 nmol/L, and 1 mmol/L) of FIPI for 12 hours. Following [32P]orthophosphate labeling, cells were treated with minimal essential medium (MEM) or MEM containing TPA (25 nmol/L; A), H2O2 (100 mmol/L; B), diamide (100 mmol/L; C), and 4-hydroxy-2-nonenal ([4-HNE] 100 mmol/L; D) for 1 hour in the presence of 0.05% (volume/ volume [vol/vol]) 1-butanol. At the end of the incubation period, [32P]phosphatidylbutanol ([32P]PBt) formed was determined. Data represent mean + standard deviation (SD) calculated from 3 independent experiments. *Significantly different at P < .05 as compared to cells treated with MEM alone. **Significantly different at P < .05 as compared to cells treated with MEM containing agonist or oxidant alone. and (iii) PLD1 threonine phosphorylation. The MAECs were N,N0 -bis(2-Mercaptoethyl)Isophthalamide Attenuates pretreated with PD98059 (0 or 10 mmol/L) for 1 hour then Hg-Induced Phosphorylation of ERK1/2 and PLD1 treated with methylmercury (0 or 10 mmol/L) for 1 hour. PD98059 significantly attenuated the methylmercury-induced Having shown in this study that Hg induced [32P]PBt forma- [32P]PBt formation to near the level in untreated control cells tion (PLD activation), upstream phosphorylation of ERK1/2, (Figure 9A). Methylmercury-induced ERK1/2 phosphorylation PLD 1 threonine phosphorylation downstream of ERK1/2 and PLD1 threonine phosphorylation as observed by SDS- phosphorylation, and NBMI attenuated the Hg-induced PAGE and Western blotting also decreased in PD98059- [ 32 P]PBt formation, next we investigated whether NBMI treated MAECs almost to the levels in untreated control cells would modulate the Hg-induced phosphorylation of ERK1/2 (Figure 9B, C). These results revealed that the ERK1/2 and PLD1 threonine phosphorylation in MAECs. Cells were upstream inhibitor attenuated the Hg-induced activation of pretreated with NBMI (0 or 25 mmol/L) for 1 hour prior to PLD1, ERK1/2 phosphorylation, and PLD1 threonine phos- being treated with methylmercury (0 or 10 mmol/L) for 1 hour. phorylation in MAECs, suggesting that ERK1/2 regulated the Treatment with NBMI caused a significant attenuation of the Hg-induced activation of PLD1 upstream. Hg-induced phosphorylation of ERK1/2 and PLD1 threonine Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 629 A B Vehicle MeHg (5 5 µM) MeHg (10 µM)) Vehicle Thimero osal (10 µM) Thimerosal (25 5 µM) NBMI (50 0 µM) NBMI (50 µM M) NBMI (50 µM) NBMI (50 µM)) + + + + NBM MI (50 µM) MeHg (55 µM) MeHg (10 µM M) M) NBMI (50 µM Thimero osal (10 µM) 5 µM) Thimerosal (25 C D Vehicle Thimerrosal (10 µM) Thimerosal (25 5 µM) V Vehicle MeHg (5 µM) MeHg (10 µM M) 50 µM) NBMI (5 NBMI (50 µM)) NBM MI (50 µM) NBMI (50 µ µM) + + + + NB BMI (50 µM) MeHg (5 µM) MeHg (10 µM)) NBMI (50 µM M) Thimerrosal (10 µM) Thimerosal (2 25 µM) Figure 7. N,N0 -bis(2-Mercaptoethyl)isopthalamide (NBMI) attenuates the mercury-induced phospholipase D1 (PLD1) and PLD2 in situ trans- location. Mouse aortic endothelial cells (MAECs) cultured on coverslips (5 ! 105 cells/35 mm dish) were pretreated with minimal essential medium (MEM) alone or MEM containing NBMI (50 mmol/L) for 1 hour then treated with MEM alone or MEM containing methylmercury (5 and 10 mmol/L) and thimerosal (10 and 25 mmol/L) for 1 hour. At the end of the incubation period, the cells were fixed, stained for PLD1 (A and B) and PLD2 (C and D), and visualized using confocal fluorescent microscopy. Each digitally captured image is a representative picture obtained from 3 independent experiments conducted under identical conditions. phosphorylation, almost to control levels, as compared to Hg-induced phosphorylation of ERK1/2 and PLD1 threonine cells treated with Hg alone (Figure 10A, B). These results phosphorylation in MAECs, suggesting the role of redox- revealed that the novel thiol stabilizer, NBMI, attenuated the regulation therein. Downloaded from ijt.sagepub.com by guest on September 17, 2012 630 International Journal of Toxicology 30(6) A B MeHg (10 µM) - + + + MeHg (1 10 µM) - + + + Time (min) 0 5 15 30 Time e (min) 0 5 1 15 30 Mol. Wt. I.B. Mol. Wt. I.B. 44 kDa Phospho-ERK1 1/2 110 kDa Phosppho-PLD1 42 kDa 44 kDa 110 kDa PLD1 ERK1/2 42 kDa (Phosphothreonine-PLD1/PLD1 0.8 1.2 * ERK Phosphorylation (Phospho-ERK/ERK * * intensity, a.u.) Phosphorylation 1 PLD1 Threonine * a.u.) u) 0.6 intensity a 0.8 intensity, 0.6 0.4 * 0.4 * 0.2 0.2 0 0 - + + + MeeHg (10 µM) - + + + MeHg (100 µM) Time (min) 0 5 15 30 Time (miin) 0 5 15 30 C D Thim merosal (25 µM) - + + sal (25 µM) - Thimeros + + Time (min) 0 5 60 T Time (min) 0 5 60 Mol. Wt. I.B B. Mo ol. Wt. I.B. 44 kDa Ph hospho-ERK1/2 11 10 kDa Phospho-PLD1 42 kDa 44 kDa 11 10 kDa PLD1 ER RK1/2 42 kDa 1.4 PLD1 Threonine Phosphorylation (Phosphothreonine-PLD1/PLD1 1 1.2 * * ERK Phosphorylation 1 intensity, a.u.) (Phospho-ERK/ERK 0.8 intensity,a.u.) 0.6 * 0.8 0.6 * 0.4 0.4 0.2 0.2 0 0 sal (10 µM) Thimeros - + + merosal (10 µM) Thim - + + e (min) Time Time (min) 0 5 60 0 5 60 Figure 8. Mercury induces phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) and phospholipase D1 (PLD1) in mouse aortic endothelial cells (MAECs). The MAECs (5 ! 105 cells/35 mm dish) were treated with minimal essential medium (MEM) alone or MEM containing methylmercury (10 mmol/L) for 5, 15, and 30 minutes (A and B) and MEM containing thimerosal (25 mmol/L) for 5 and 60 minutes (C and D). Following treatment, proteins in cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with phospho-ERK1/2-specific, ERK1/2-specific, phosphotheonine-PLD1-specific, and PLD1-specific monoclonal antibodies. The intensity of the protein bands were digitally determined as described in Materials and Methods. *Significantly different at P < .05 as compared to cells treated with MEM alone. N,N0 -bis(2-Mercaptoethyl)Isophthalamide Protects PLD activation in MAECs. In addition, we hypothesized Against Hg- and Oxidant-Induced GSH Loss that the loss of intracellular soluble thiols could be protected by the novel thiol-redox antioxidant, NBMI. To determine this Our earlier studies have demonstrated that the oxidant- and Hg- relationship, MAECs were pretreated with NBMI (0 or induced PLD activation in ECs is thiol redox regulated and is 50 mmol/L) for 1 hour prior to treatment with Hg (methylmer- associated with the loss of thiols including GSH.7,26 Therefore, cury, 10 mmol/L or thimerosal, 25 mmol/L) or oxidant (H2O2, here we hypothesized that Hg and oxidants would cause the 100 mmol/L or diamide, 250 mmol/L) for 1 hour. Both Hg and depletion of intracellular soluble thiols concomitant with the Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 631 PD (10 µM) - + - + MeHg (10 µM) - - + + Mol. Wt. I.B. A B 44 kDa Phospho-ERK1/2 42 kDa 2500 Vehicle 44 kDa PD98059 (10 µM) * 42 kDa ERK1/2 [32P] PBt Formed (DPM/Dish) (Phospho-ERK/ERK intensity, a.u.) 1.2 2000 1 * ERK Phosphorylation 1500 ** 0.8 ** 1000 0.6 * 0.4 500 0.2 0 0 Control MeHg (10 µM) PD (10 µM) - + - + MeHg (10 µM) - - + + PD (10 µM) - + - + MeHg (10 µM) - - + + C Mol. Wt. I.B. 110 kDa Phospho-PLD1 110 kDa PLD1 (Phosphothreonine PLD1/PLD1, a.u.) 0.5 * 0.4 PLD1 Phosphorylation 0.3 0.2 ** * 0.1 0 PD (10 µM) - + - + MeHg (10 µM) - - + + Figure 9. Extracellular-regulated kinase 1/2 (ERK1/2) upstream inhibitor, PD98059, attenuates the mercury-induced phospholipase D (PLD) activation, ERK1/2 phosphorylation, and PLD1 phosphorylation in mouse aortic endothelial cells (MAECs). The MAECs (5 ! 105 cells/ 35 mm dish) were labeled with [32P]orthophosphate in phosphate-free Dulbecco-modified Eagle medium (DMEM) for 12 hours. Following [32P]orthophosphate labeling, cells were pretreated with minimal essential medium (MEM) alone or MEM containing ERK1/2 upstream inhibitor, PD98059 (10 mmol/L), for 1 hour. Following pretreatment, cells were treated with MEM alone or MEM containing methylmercury (10 mmol/L) for 1 hour. At the end of the incubation period, [32P]phosphatidylbutanol ([32P]PBt) formed was determined (A). B, MAECs (5 ! 105 cells/35 mm dish) pretreated with MEM alone or MEM containing PD98059 (10 mmol/L) for 1 hour were treated with MEM alone or methylmercury (10 mmol/L) for 1 hour. Following treatment, proteins in cell lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with phospho-ERK1/2-specific, ERK1/2-specific, (C) phosphotheo- nine-PLD1-specific, and PLD1-specific monoclonal antibodies. The intensity of the protein bands were digitally determined as described in Materials and Methods. Data represent mean + standard deviation (SD) calculated from 3 independent experiments. *Significantly different at P < .05 as compared to cells treated with MEM alone. **Significantly different at P < .05 as compared to cells treated with MEM containing mercury alone. oxidant treatment demonstrated significant loss of GSH in levels measured in untreated control cells. These results MAECs. N,N0 -bis(2-Mercaptoethyl)isophthalamide offered demonstrated that the novel thiol-redox antioxidant, NBMI, significant attenuation of the Hg- and oxidant-induced loss of protected the Hg- and oxidant-induced loss of the intracellular GSH in cells, restoring the levels of GSH in treated cells to the soluble thiol, GSH (Figure 11). Downloaded from ijt.sagepub.com by guest on September 17, 2012 632 International Journal of Toxicology 30(6) 8 A - + - + Vehicle NBMI (50 µM) NBMI (25 µM) 7 ** MeHg (10 µM) ** - - + + 6 GSH (µM/106 cells) Mol. Wt. I.B. † 5 ** 44 kDa Phospho-ERK1/2 42 kDa 4 44 kDa ERK1/2 3 ** 42 kDa * 1.8 2 1.6 * 1 * * ERK Phosphorylation (Phospho-ERK/ERK 1.4 * intensity, a.u.) 0 1.2 Control MeHg (10 µM) Thimerosal (25 µM) H2O2 (100 µM) Diamide (250 µM) 1 0.8 0.6 Figure 11. N,N0 -bis(2-Mercaptoethyl)isopthalamide (NBMI) pro- 0.4 tects against the mercury- and oxidant-induced glutathione (GSH) loss. Mouse aortic endothelial cells ([MAECs] 5 ! 105 cells/35 mm 0.2 * ** dish) pretreated with minimal essential medium (MEM) alone or 0 - + - MEM containing NBMI (50 mmol/L) for 1 hour were treated with NBMI (25 µM) + MeHg (10 µM) MEM alone or MEM containing methylmercury (10 mmol/L) or thi- - - + + merosal (25 mmol/L) or H2O2 (100 mmol/L) or diamide (250 mmol/ L). At the end of the incubation period, the intracellular soluble thiol (glutathione [GSH]) concentrations were determined. Data B represent mean + standard deviation (SD) calculated from 3 inde- NBMI (25 µM) - + - + pendent experiments. *Significantly different at P < .05 as compared MeHg (10 µM) - - + + to cells treated with MEM alone. **Significantly different at P < .05 Mol. I.B. as compared to cells treated with MEM containing mercury or oxi- 110 kDa Phospho-PLD1 dant alone. 110 kDa PLD1 Both FIPI and NBMI Protect Against Hg- and 0.7 Oxidant-Induced Cytotoxicity PLD1 Threonine Phosphorylation * (Phosphothreonine PLD1/PLD1 0.6 Phospholipase D-mediated PA signaling has been implicated 0.5 in several signaling pathways and the role of PA has been intensity, a.u.) * ** 0.4 previously reported in the oxidant-induced cytotoxicity in 0.3 ECs.39 Hence, we hypothesized that Hg and oxidants which 0.2 induced significant activation of PLD would also cause cyto- 0.1 toxicity in MAECs. Furthermore, we hypothesized that NBMI and FIPI, which attenuated the Hg- and oxidant-induced PLD 0 activation, would offer protection against the Hg- and NBMI (25 µM) - + - + oxidant-induced cytotoxicity in MAECs. In order to investi- MeHg (10 µM) - - + + gate this hypothesis, MAECs were pretreated with FIPI (0, 250 nmol/L, and 1 mmol/L for 12 hours) or NBMI (0 or Figure 10. N,N0 -bis(2-Mercaptoethyl)isopthalamide (NBMI) attenuates 50 mmol/L for 1 hour) prior to treatment with Hg (methylmer- the mercury-induced phosphorylation of extracellular-regulated kinase cury, 10 mmol/L, or thimerosal, 25 mmol/L) or oxidant (dia- 1/2 (ERK1/2) and phospholipase D1 (PLD1) in mouse aortic endothelial mide, 250 mmol/L) for 1 hour, and cytotoxicity (LDH release cells (MAECs). The MAECs (5 ! 105 cells/35 mm dish) pretreated with minimal essential medium (MEM) alone or MEM containing NBMI and MTT reduction) was measured at the end of treatment. (25 mmol/L) for 1 hour were treated with MEM alone or MEM containing Both Hg and diamide caused significant cytotoxicity in methylmercury (10 mmol/L) for 1 hour. Following treatment, proteins in MAECs as compared with untreated control cells. N,N 0 - cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel bis(2-Mercaptoethyl)isophthalamide offered significant electrophoresis (SDS-PAGE) and Western blotting with (A) phospho- attenuation of the Hg- and diamide-induced LDH release by ERK1/2-specific, ERK1/2-specific, (B) phosphotheonine-PLD1-specific MAECs (Figure 12A, B). Also, FIPI significantly attenuated and PLD1-specific monoclonal antibodies. The intensity of the protein Hg- and diamide-induced inhibition of MTT reduction in bands were digitally determined as described in Materials and Methods section. Data represent mean + standard deviation (SD) calculated from MAECs (Figure 12C, D). Thus, these results revealed that the 3 independent experiments. *Significantly different at P < .05 as com- novel thiol-redox antioxidant, NBMI, and the novel PLD- pared to cells treated with MEM alone. **Significantly different at P < .05 specific inhibitor, FIPI, effectively protected against the Hg- as compared to cells treated with MEM containing mercury alone. and oxidant-induced cytotoxicity in MAECs. Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 633 A B 0.12 Vehicle 0.9 Δ Absorbance (490nm - 690nm) Δ Absorbance (570nm - 690nm) Vehicle 0.1 * FIPI (1 µM) 0.8 * 0.7 FIPI (250 nM) 0.08 * 0.6 ** ** 0.5 * ** 0.06 ** 0.4 ** 0.04 ** * 0.3 0.2 0.02 * 0.1 * * 0 0 Control MeHg (10 µM) Thimerosal (25 µM) Diamide (250 µM) Control MeHg (10 µM) Thimerosal (25 µM) Diamide (250 µM) C D 0.25 0.8 Vehicle * Δ Absorbance (490nm - 690nm) Vehicle Δ Absorbance (570nm - 690nm) NBMI (50 µM) NBMI (50 µM) 0.7 ** ** 0.2 0.6 ** 0.5 0.15 * 0.4 * 0.1 * 0.3 ** ** ** 0.2 0.05 * * * 0.1 0 0 Control MeHg (10 µM) Thimerosal (25 µM) Diamide (250 µM) Control MeHg (10 µM) Thimerosal (25 µM) Diamide (250 µM) Figure 12. 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI) and N,N0 -bis(2-mercaptoethyl)isopthalamide (NBMI) protect against the mercury- and oxidant-induced cytotoxicity in mouse aortic endothelial cells (MAECs). The MAECs (2.5 ! 105 cells/17.5 mm dish) were pretreated with minimal essential medium (MEM) alone, MEM containing FIPI (10 mmol/L) for 12 hours, or MEM containing NBMI (50 mmol/L) for 1 hour. Pretreated cells were then treated with MEM alone or MEM containing methylmercury (10 mmol/L) or thimerosal (25 mmol/L) or diamide (250 mmol/L) for 1 hour. At the end of the incubation period, release of lactate dehydrogenase (LDH) into the medium (A and C) and decrease in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction (B and D; as an index of cytotoxicity) were determined spectro- photometrically. Data represent mean + standard deviation (SD) calculated from 3 independent experiments. *Significantly different at P < .05 as compared to cells treated with MEM alone. **Significantly different at P < .05 as compared to cells treated with MEM containing mercury or oxidant alone. Discussion levels which were restored by NBMI; and (vi) NBMI and FIPI offered protection against the Hg- and oxidant-induced cyto- In the current study, we investigated the thiol-redox-mediated toxicity in MAECs. This is the first observation which demon- and ERK-regulated PLD signaling as a mechanism of Hg cyto- strated that thiol-redox-dependent and ERK1/2-regulated PLD toxicity in MAECs utilizing the novel lipid-soluble, thiol-redox activation led to the Hg-induced vascular EC cytotoxicity antioxidant and heavy metal chelator, NBMI, and the novel through bioactive lipid signal mediators which was protected PLD-specific inhibitor, FIPI. The results revealed that (i) all by the novel thiol-redox antioxidant and heavy metal chelator, the 3 chosen forms of Hg including the inorganic form of Hg NBMI, and novel PLD-specific inhibitor, FIPI (Figure 13). (mercury(II) chloride), environmental organic form of Hg Phospholipases catalyze the hydrolysis of membrane (methylmercury), and the pharmaceutical organic form (thi- phospholipids, leading to the generation of bioactive lipid sec- merosal) induced PLD activation in a dose- and time- ond messengers which play crucial roles in cellular signaling dependent manner which was completely attenuated by NBMI cascades.25,27 Phospholipase D, one such phospholipid hydro- and FIPI; (ii) NBMI and FIPI also attenuated the oxidant- lase and a lipid signaling enzyme, is ubiquitously present in induced PLD activation; (iii) Hg induced in situ translocation all mammalian cells that preferentially acts on phosphatidyl- of PLD1 and PLD2 that was attenuated by NBMI; (iv) Hg choline (PC) generating PA and choline. Phosphatidic acid is induced upstream phosphorylation of ERK1/2 which caused further metabolized into either 1,2-DAG by phosphatidate the downstream threonine phosphorylation of PLD1 which was phosphohydrolase or to LPA by phospholipase A1/A2.25,27 attenuated by NBMI; (v) Hg depleted the intracellular GSH Agonist-mediated activation of PLD regulates signal Downloaded from ijt.sagepub.com by guest on September 17, 2012 634 International Journal of Toxicology 30(6) classic thiol-redox antioxidant, NAC, and the thiol-chelating drug, DMSA, also attenuated the Hg-induced PLD activation in MAECs. This is also supported by our earlier reported finding that the Hg-induced PLD activation in BPAECs is attenuated by both NAC and DMSA, which are both water-soluble drugs.7 However, the novel thiol-redox antioxidant, NBMI, used in this study is hydrophobic and lipid-soluble which might have ren- dered the compound to exhibit its actions more effectively in the hydrophobic sites of the phospholipid bilayer of the cell membrane such as attenuating the Hg-induced PLD activation in MAECs. This study, thus clearly established that the Hg- induced PLD activation and the generation of the lipid signal mediator, PA, were thiol-redox regulated. This was further confirmed in the current study by the observations that the PLD activation in MAECs induced by oxidants such as 4-HNE (a lipid peroxidation-derived reactive carbonyl) and DPV (an oxi- dant and thiol-redox perturbing agent) was also attenuated by NBMI. Oxidant-induced PLD activation in ECs through ROS generation and thiol-redox dysregulation and attenuation of the Figure 13. Proposed mechanism of protection by N,N0 -bis(2-mercap- enzyme activation by thiol-redox enhancer/stabilizer, NAC, toethyl)isopthalamide (NBMI) and 5-fluoro-2-indolyl des- has been documented.25,27,39,41 Diamide, a thiol-depleting chlorohalopemide (FIPI) against mercury-induced cytotoxicity in aortic agent, has been reported to cause PLD activation in vascular endothelial cells (ECs) through the attenuation of thiol-redox–regulated ECs that is attenuated by the thiol-protecting agents such as and extracellular-regulated kinase 1/2 (ERK1/2)–mediated activation of NAC and mercaptopropionylglycine.26 Translocation and relo- phospholipase D (PLD) and bioactive lipid signal mediator formation. calization of PLD isoenzymes in situ have been shown to be associated with the oxidant-induced PLD activation in vascu- transduction in mammalian cells.25 Phospholipase D-generated lar ECs.25 Along those lines, the current study also demon- lipid mediators including PA and LPA are involved in critical strated that the Hg-induced in situ translocation of PLD1 and cellular functions such as mitogenesis in fibroblasts, oxidative PLD2 isoenzymes in MAECs was also attenuated by the thiol- burst in neutrophils, elevation of intracellular calcium levels, and redox stabilizer, NBMI. Our findings clearly revealed that activation of protein kinases.25 The 2 major isoenzymes of PLD, NBMI was not only effective in attenuating the Hg-induced PLD1, and PLD2, have been well characterized and cloned in PLD activation but was also effective in attenuating the mammalian cells and are selectively activated by different oxidant-induced enzyme activation in MAECs, suggesting its cofactors such as Arf, Rho, and Cdc42.41 Phosphatidylinositol effectiveness in restoring thiol-redox balance as Hg, 4-HNE, 4,5-bisphosphate (PIP2), a lipid mediator, has been shown to and DPV are known to cause PLD activation in ECs through activate both PLD1 and PLD2 in vitro.41 thiol-redox alteration.7,26,44 The current study revealed that all the 3 chosen forms of Hg Others and we have reported that oxidants induce PLD acti- activated PLD and induced the formation of PBt (an index of vation in vascular ECs, vascular smooth muscle cells, and PA formation) in MAECs in a dose- and time-dependent fash- fibroblasts,25,41 which is attenuated by antioxidants. It is impor- ion. In addition, the study also demonstrated that the Hg- tant to mention here that the oxidant-induced PLD activation is induced PLD activation was regulated upstream by the thiol also regulated by the upstream activation of certain key protein redox in MAECs as supported by our finding that Hg caused kinases, leading to the phosphorylation of PLD in vascular the loss of GSH which was restored by the novel thiol-redox ECs. Hydrogen peroxide-induced PLD activation in ECs has agent, NBMI, leading to the attenuation of PLD activation in been reported to be regulated upstream by the activation of the cells. Our current finding is also supported by our earlier protein tyrosine kinases. 45 Diperoxovanadate has been report that Hg as mercury (II) chloride, methylmercury, and observed to stimulate PLD activity in vascular ECs through the thimerosal activates PLD in the BPAECs which is regulated by upstream activation and regulation of Src kinase and p38 thiol redox and ROS.7 However, in the current study, we uti- mitogen-activated protein kinase (p38 MAPK).41,42 Vitamin lized the MAECs as our in vitro vascular EC model which C, a pro-oxidant, has been reported to activate PLD in lung represents the aortic endothelium and is different from our microvascular ECs through the upstream thiol-redox regulation previously utilized BPAEC model that originates from the pul- and activation of ERK1/2 and p38 MAPK.25 These studies monary arteries. The rationale for using the MAECs in the have clearly established that the phosphorylation of PLD iso- current study as the in vitro model was to justify the systemic enzymes is an essential requirement upstream of activation of vascular endothelium as the target for Hg adverse effects, espe- PLD by oxidants in vascular ECs. The MAPKs, the stress- cially the activation of the lipid signaling cascades such as activated protein kinases, are broadly divided into 3 classes, PLD. Our study also revealed that in addition to NBMI, the namely (i) ERK, (ii) c-Jun N-terminal kinase (JNK), and (iii) Downloaded from ijt.sagepub.com by guest on September 17, 2012 Secor et al. 635 p38 MAPK, all of which take part in diverse cellular signaling heterotrimeric and small molecular weight G proteins, their cascades, upon activation through phosphorylation of role in the Hg-induced PLD activation in MAECs was not ruled both threonine and tyrosine residues by dual-specific serine– out, considering the fact that Hg has been shown to perturb the threonine MAPK kinases.25 Mercury has been shown to acti- thiol-redox status and induced ROS in vascular ECs.7,48 vate MAPKs in model systems such as the H4IIE cells, renal In the current study, we took advantage of the availability of cortical slices, and macrophages.43,46,47 As the mechanism of the novel PLD-specific inhibitor, FIPI, to not only confirm the the Hg-induced PLD activation in vascular ECs has not been PLD activation induced by Hg but also to establish for the first established so far, here, we investigated whether ERK1/2 time its specificity to cause inhibition of the agonist- and would be involved in the upstream activation of PLD in oxidant-induced PLD activation in ECs. Our study demon- MAECs through threonine phosphorylation of the enzyme. Our strated that FIPI effectively attenuated the Hg-, oxidant-, and current results are in accord with the reports made by others by TPA-induced PLD activation in MAECs. This finding was revealing that ERK1/2 upstream-specific inhibitor (PD98059) further supported by our earlier report that bleomycin- not only attenuated the Hg-induced PLD activation but also induced and thiol-redox-regulated PLD activation in lung attenuated the Hg-induced upstream ERK1/2 phosphorylation microvascular ECs is attenuated by FIPI.39 Mercury is reported and PLD1 threonine phosphorylation in MAECs, suggesting to cause PLD activation through thiol-redox dysregulation and that ERK1/2 was the upstream regulator of Hg-induced PLD ROS.7 Oxidants are shown to activate PLD through thiol-redox activation. In this context, we were able to utilize the only alteration, protein tyrosine kinase activation, and MAPK sig- commercially available antiphosphothreonine PLD1 antibody naling in ECs.25,26,41,42 TPA is an established agonist to cause to determine the ERK1/2-mediated threonine phosphorylation activation of PLD through PKC signaling in ECs.49 Therefore, of PLD1 in the Hg-treated MAECs by SDS-PAGE and Western our current study established that FIPI effectively inhibited blotting. Due to the nonavailability of antiphosphothreonine PLD activation in MAECs, irrespective of agonist-mediated PLD2 antibody, we were unable to determine the ERK1/2- induction and upstream signaling regulation involving thiol mediated threonine phosphorylation of PLD2 in the Hg- redox, ROS, protein tyrosine kinases, MAPK, and PKC and treated MAECs. However, threonine phosphorylation of PLD2 further suggested that FIPI can be used as a PLD-specific inhi- induced by Hg in MAECs could be possible and was not ruled bitor in studies aimed at PLD-mediated bioactive cellular lipid out in our current study. More noticeably, the upstream phos- signaling cascades. Also, the current study clearly revealed that phorylation of ERK1/2 and the downstream threonine phos- FIPI protected against the Hg-induced cytotoxicity in MAECs phorylation of PLD1 and activation of PLD induced by Hg in suggesting the role of PLD therein. This finding was supported MAECs were effectively attenuated by the novel thiol-redox by our earlier report that the bleomycin-induced cytotoxicity stabilizer, NBMI, further strengthening the notion that thiol through bioactive lipid (PA) formation in lung microvascular redox played a critical role in regulating the Hg-induced PLD ECs is protected by the PLD-specific inhibitor, FIPI. From our activation through upstream ERK1/2-mediated threonine phos- current study, it could be surmised that the bioactive lipids phorylation of the enzyme. Nevertheless, the exact mechanism generated by PLD including PA, LPA, and DAG would have of the Hg-induced ERK1/2 activation and the resultant site- caused cytotoxicity of Hg in MAECs. specific threonine phosphorylation in PLD1 in ECs warrant More striking observation of the current study was the pro- further investigation. tection of the Hg-induced cytotoxicity by the novel thiol-redox The regulation of the agonist-induced PLD activation is antioxidant, NBMI, in MAECs through restoring the loss of apparently complex involving PKC, heterotrimeric G proteins, GSH and attenuating the ERK1/2-mediated PLD signaling. It small molecular weight G proteins, and protein tyrosine has been established that Hg preferentially reacts with cellular kinases/protein tyrosine phosphatases.25 Hormones, growth thiols, and GSH being the most critical intracellular soluble factors, neurotransmitters, cytokines, and ROS induce PLD thiol antioxidant is depleted during Hg toxicity.50 Association activation in a wide variety of mammalian cell systems and of GSH depletion during thimerosal neurotoxicity and its pro- tissues.25,41 Agonist-specific and cell-specific mechanisms, tection by GSH precursors (GSH ethyl ester and NAC) have through signaling modulation by PKC, intracellular calcium, been documented.50 Low-molecular-weight thiols such as L- heterotrimeric G proteins, protein tyrosine kinases/phospha- cysteine, DL-homocysteine, L-methionine, and NAC have been tases, small molecular weight G proteins or Rho GTPase class, shown to protect against the thimerosal-induced neurotoxicity and MAPKs, regulate the agonist-induced PLD activation in in cerebellar granule cells.51 Thiol chelators including NAC cellular systems. Oxidant-specific signaling mechanisms reg- and dithiothreitol have been shown to protect against the ulate the oxidant-induced PLD activation in cells through PKC dimethylmercury-induced toxicity in rats.52 Common metal or protein tyrosine kinases or MAPKs involving thiol chelators such as calcium disodium ethylenediaminetetraace- redox. 25,39 Protein tyrosine kinases and PKC have been tate (CaNa2EDTA), D-penicillamine, 2,3-dimercaptopropane- reported to regulate the H2O2-induced PLD activation in Swiss 1-sulfonate (DMPS), and DMSA have been reported to offer 3T3 fibroblasts.25,41 Earlier, we have also shown the involve- protection against the Hg-induced neurotoxicity in cortical ment of intracellular calcium in the Hg-induced PLD activation cells. 53 These reports have established that the thiol- in vascular ECs.22 Although the current study did not examine protective compounds offer protection against Hg-induced the involvement of PKC, protein tyrosine kinases, and toxicity and further support our current findings that NBMI Downloaded from ijt.sagepub.com by guest on September 17, 2012 636 International Journal of Toxicology 30(6) offered protection against Hg cytotoxicity in MAECs through However, this research project had not been funded or had not been stabilization of thiol-redox-mediated ERK1/2 and PLD signal- supported or carried out in part or full by CTI Science in any way. ing. The results of the current study also demonstrated that Neither Dr Haley nor CTI Science had any influence or control on this DMSA, the water-soluble thiol chelator, was equally effective research project. This research project was entirely initiated and super- vised by Narasimham L. Parinandi at the Ohio State University, as NBMI, the lipid-soluble thiol antioxidant and chelator in Columbus, OH, USA, with the compound, NBMI synthesized by attenuating the Hg-induced PLD activation in MAECs. How- Niladri Gupta (a graduate student of Boyd E. Haley) in the Department ever, it should be emphasized that DMSA is only a heavy metal of Chemistry at the University of Kentucky, Lexington, KY, USA, chelator, whereas NBMI is both a heavy metal chelator and a under the supervision of Dr Haley. Boyd E. Haley is Chair of the thiol antioxidant. Moreover, the present study was only aimed Scientific Advisory Committee of the International Academy of Oral at investigating the efficacy of the novel lipid-soluble thiol Medicine and Toxicology (IAOMT), who advises the IAOMT Board antioxidant and heavy metal chelator, NBMI. Although NAC regarding scientific matters, but he does not have any control over and GSH have been used for treating Hg toxicity, caution has to IAOMT in their grant funding decisions. The IAOMT award has been be exercised while using these thiols for treatment of Hg toxi- given exclusively to Narasimham L. Parinandi to conduct research city, as the low-molecular-weight thiols containing cysteine are without any influence or control of Boyd E. Haley on this research identified to be critical in the transport and body disposition of project. Overall, there are no conflicts of interest. the metal regulated by molecular mimicry.33 In addition, the low-molecular-weight thiol-protective compounds such as Funding NAC and GSH undergo autoxidation/oxidative attack and also The author(s) disclosed receipt of the following financial support for act as pro-oxidants further contributing to oxidative stress, the research, authorship, and/or publication of this article: the Dorothy which should be also taken into serious consideration while M. Davis Heart and Lung Research Institute and the Division of using them for treatment of Hg toxicity. Pulmonary, Allergy, Critical Care, and Sleep Medicine of the Ohio Structurally, NBMI possesses dicarboxybenzoate moiety as State University College of Medicine, the National Institute of Health that occurs naturally in fruits that is bound to 2 cysteamines. (HL093463), the International Academy of Oral Medicine and Therefore, it is reasonable to expect that NBMI could act as Toxicology (IAOMT), and the Alan D. Clark, MD, Memorial Research both a Hg chelator and free radical scavenger. The ability of Fund. NBMI to complex with trace heavy metals including Hg has been reported.54 Hence, the cytoprotective action of NBMI against the Hg-induced cytotoxicity through thiol-redox regu- References lation, ERK1/2 activation, and PLD activation as observed in 1. Clarkson TW, Magos L, Myers GJ. The toxicology of mercury- the current investigation could have been operated by the abil- current exposures and clinical manifestations. N Engl J Med. ity of NBMI in stabilizing/protecting the cellular thiol redox, 2003;349(18):1731-1737. attenuating oxidative stress, and sequestering Hg. In addition, 2. Bolger PM, Schwetz BA. Mercury and health. 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Novel Lipid-Soluble Thiol-Redox Antioxidant and Heavy Metal Chelator, N,N'-bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aorti...

Novel Lipid-Soluble Thiol-Redox Antioxidant and Heavy Metal Chelator, N,N'-bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aorti...

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