J BIOCHEM MOLECULAR TOXICOLOGYVolume 22, Number 6, 2008

Isocyanates Induces DNA Damage, Apoptosis,Oxidative Stress, and Inflammation in Cultured HumanLymphocytesPradyumna Kumar Mishra,1 Hariom Panwar,1 Arpit Bhargava,1 Venkata RaghuramGorantla,1 Subodh Kumar Jain,2 Smita Banerjee,2 and Kewal Krishan Maudar1

1Bhopal Memorial Hospital & Research Centre, Raisen Bypass Road, Bhopal 462 038, India; E-mail: pkm 8bh@yahoo.co.uk2Department of Biotechnology, H. S. Gour University, Sagar 470 003, India

Received 26 May 2008; revised 11 August 2008; accepted 23 August 2008

ABSTRACT: Isocyanates, a group of low molecularweight aromatic and aliphatic compounds containingthe isocyanate group (−NCO), are important raw ma-terials with diverse industrial applications; however,pathophysiological implications resulting from occu-pational and accidental exposures of these compoundsare hitherto unknown. Although preliminary evidenceavailable in the literature suggests that isocyanates andtheir derivatives may have deleterious health effectsincluding immunotoxicity, but molecular mechanismsunderlying such an effect have never been addressed.The present study was carried out to assess the im-munotoxic response of methyl isocyanate (MIC) oncultured human lymphocytes isolated from healthyhuman volunteers. Studies were conducted to eval-uate both dose-dependent and time-course response(n = 3), using N-succinimidyl N-methylcarbamate, asurrogate chemical substitute to MIC. Evaluation ofDNA damage by ataxia telangiectasia mutated (ATM)and γH2AX protein phosphorylation states; measureof apoptotic index through annexin-V/PI assay, apop-totic DNA ladder assay, and mitochondrial depolariza-tion; induction of oxidative stress by CM-H2DCFDAand formation of 8-hydroxy-2′deoxy guanosine; lev-els of antioxidant defense system enzyme glutathionereductase; and multiplex cytometric bead array anal-ysis to quantify the secreted levels of inflammatorycytokines, interleukin-8, interleukin-1ββ, interleukin-6,interleukin-10, tumor necrosis factor, and interleukin-12p70 parameters were carried out. The results of thestudy showed a dose- and time-dependent response,providing evidence to hitherto unknown molecularmechanisms of immunotoxic consequences of iso-cyanate exposure at a genomic level. We anticipatethese data along with other studies reported in theliterature would help to design better approaches in

Correspondence to: Pradyumna Kumar Mishra.c© 2008 Wiley Periodicals, Inc.

risk assessment of occupational and accidental expo-sure to isocyanates. C© 2008 Wiley Periodicals, Inc. JBiochem Mol Toxicol 22:429–440, 2008; Published on-line in Wiley InterScience (www.interscience.wiley.com).DOI 10:1002/jbt.20260

KEYWORDS: Immunotoxicity; Isocyanates; DNA Dam-age; Oxidative Stress; Apoptosis; Methyl Isocyanate

INTRODUCTION

Global analysis of immune response to toxic ex-posures has been largely documented in the past twodecades, but it has not been easy to delineate theseeffects on immune system. Evidence from different co-hort studies suggests that immune system is a possibletarget of toxicity after exposure to a wide array of in-dustrial and environmental chemicals [1]. Even thoughour immunity, one way or other, is influenced by theenvironment, reduction in the number of immunocom-petent cells or alterations in function, selection, anddifferentiation of lymphocytes following occupationalor accidental exposures might have detrimental effects[2].

Perturbations following exposure to immunotoxicchemicals might exert a strong impact on processes ata genomic level. In response to stress, ataxia telang-iectasia mutated (ATM) kinase, a phosphoinositide-3-kinase-like kinases (PIKKs), plays an essential rolein maintaining genome integrity by coordinatingand DNA damage repair and apoptosis, and ATMgets autophosphorylated at Serine 1981 residue, thusactivating the downstream signaling molecules of thecascade to effectively repair the damage [3]. An impor-tant substrate for the ATM kinase cascade is H2AX,

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a variant isoform of the histone H2A protein family,which becomes extensively phosphorylated on serine139 residue in response to damage to DNA, thereby,forming large distinct nuclear γ-H2AX (γH2AX) fociand appears during apoptosis concurrently with theinitial appearance of high molecular weight DNAfragments, facilitating the packaging of fragmentedDNA into apoptotic bodies [4]. One of the character-istic features of apoptosis is also the mitochondrialdepolarization [5], which in succession contributesfor the enhanced generation of intracellular reactiveoxygen species (ROS) predominantly involving super-oxide anion (O−

2 ), hydrogen peroxide (H2O2), and thehydroxyl radicals (•OH) [6]. 2′, 7′, Dichlorodihydroflu-orescein diacetate (CM-H2DCFDA) is an oxidation-sensitive fluorescent dye commonly employed for de-tecting cellular H2O2 [7]. Upon toxic insult, inevitableoxidative DNA damage manifested as a consequenceof cellular metabolism, produces increased levels of8-oxo-2′deoxyguanosine lesion, which in turn has beenascribed much importance, largely to the detrimentof other base lesions [8]. Several antioxidant systemssuch as glutathione (GSH) limit the damage caused byROS and protect cells from oxidative stress-inducedapoptosis [9], whereas, in contrast, depletion of GSHis associated with an increased proportion of cells un-dergoing apoptosis [10]. Glutathione reductase (GR),a homodimeric flavoprotein disulfide oxidoreductase,plays an indirect but essential role in the prevention ofoxidative damage within the cell by helping to main-tain appropriate levels of intracellular reduced GSH[11]. In addition, oxidative stress is known to induceinflammation in peripheral systems of the body [12].

Isocyanates, a group of low molecular weight aro-matic and aliphatic compounds containing the func-tional isocyanate group (-NCO), are important raw ma-terials with diverse industrial applications; however,pathophysiological implications resulting from occu-pational and accidental exposures of these compoundsare hitherto unknown. Although preliminary evidenceavailable in the literature suggests that isocyanates andtheir derivatives may have deleterious health effects in-cluding implications on immune system [13,14], molec-ular mechanisms underlying such an effect have neverbeen addressed. Methyl isocyanate (MIC), a reactive in-dustrial byproduct is one of the most toxic isocyanatesand is known to exert detrimental effects on numerousorgan systems and immunity [15]. In vitro studies car-ried out on MIC and its reaction products have shownevidence of mutagenicity evoking probable clastogenicactivity [16]. MIC is also known to react with exo-cyclic amino group of dNTP’s to produce carbamoy-lated products [17,18], producing DNA cross-links inturn contributing to cytotoxicity [19]. These data impli-cate that occupational and accidental exposure to MIC

might possibly increase an individual’s susceptibilityby eliciting hypersensitivity reactions, causing autoim-munity, or immune suppression.

The goal of the present study was to evalu-ate the immunotoxic potential of MIC on peripheralblood lymphocytes following in vitro exposure to N-succinimidyl N-methylcarbamate. The study was car-ried out in peripheral blood lymphocytes isolated fromhealthy individuals. Evaluation of the activated DNAdamage response pathways of ATM and γH2AX pro-tein phosphorylation states; measure of apoptotic indexthrough annexin-V/PI assay, apoptotic DNA ladderassay, and mitochondrial depolarization; induction ofoxidative stress by CM-H2DCFDA and formation of 8-hydroxy-2′deoxy guanosine; levels of antioxidant de-fense system enzyme GR; and multiplex cytometricbead array analysis to quantify the secreted levels ofinflammatory cytokines, interleukin-8, interleukin-1β,interleukin-6, interleukin-10, tumor necrosis factor, andinterleukin-12p70 parameters were carried out.

MATERIALS AND METHODS

Subject Selection

The study was approved by the Institutional Re-view Board, Bhopal Memorial Hospital and ResearchCentre, Bhopal (India). Informed consent was obtainedfrom human subjects included in the study. The bloodwas considered healthy following routine laboratoryanalysis.

Reagents

N-succinimidyl N-methylcarbamate, a surrogatechemical equivalent to MIC [20] [CAS No. 18342–66-0] Sigma Aldrich Laboratories (St. Louis, MO) dis-solved in 2-mM DMSO with final concentration of0.005 μM was used for investigations. Phytohemagglu-tinin and RPMI growth medium were procured fromGibco-BRL Invitrogen Co. (Carlsbad, CA). Isolation oflymphocytes from peripheral blood was performedusing Lymphosep® from MP Biomedicals (Solon, OH).For assessment of DNA damage response, immunolabeling of ATM and γH2AX was done, using anti-bodies from Abcam (Cambridge, UK) and Calbiochem(Nottingham, UK), respectively. To quantify apoptosis,Annexin-V FLUOS staining kit and Apoptotic DNALadder kit from Roche Applied Sciences, Mannheim,Germany were used. Mitochondrial depolarizationwas studied by Mitochondrial Membrane Potential De-tection kit from BDTM Biosciences, San Diego, CA. De-termination of ROS was performed by CM-H2DCFDAfrom Molecular Probes, Invitrogen Co. (Carlsbad, CA).

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Formation of 8-hydroxy-2′deoxy guanosine (8-oxo-dG) and depletion of GR, markers of oxidative stresswere evaluated by using ELISA kit from Trevigen Inc.(Gaithersburg, MD). Analysis of secreted levels of in-flammatory cytokines was performed using BDTM Mul-tiplex Cytometric Bead Array (CBA) Human Inflamma-tion kit from BDTM Biosciences (San Diego, CA).

Study Design

Studies were conducted in two sections: dose-dependent and time-course kinetics (n = 3). Dose-dependent response of N-succinimidyl N-methyl-carbamate on human lymphocytes was conducted withconcentration ranging from 0.1 to 100 μg, whereas time-course experiments were performed with constant con-centration of 5 μg at time intervals ranging from 15 minto 24 h.

Lymphocyte Isolation and Culture

Heparinized venous blood of 20 mL was col-lected and isolation of lymphocytes was performedusing Lymphosep® followed by washing in phos-phate buffered saline (PBS, pH 7.4). Lymphocytes, thusseparated, were examined for viability using trypanblue dye exclusion test and counted by Neubaer’shemocytometer. 1 × 106 cells were cultured in 35 mmpetridishes (Nunc Nalgene, Rochester, NY, USA) with2-mL RPMI 1640 media (pH 7.4) supplemented with10-mM/L L-glutamine, 24-mM/L NaHCO3, 10-mM/LHepes, 10,000-U/mL penicillin, and 10,000-μg/mLstreptomycin. Cultured lymphocytes were mitogeni-cally stimulated with the addition of 0.2 mL of phy-tohemagglutinin, followed by incubation of culturedishes at 37◦C (Thermo Electron Co., Waltham, MA,USA) in 5% CO2 atmosphere with 95% relative humid-ity for 24 h.

DNA Damage Response

Measurement of ATM and γH2AX Phosphoryla-tion States through Flow Cytometry

For the quantitation and kinetics measurementof ATM and γH2AX, indirect immunofluorescencemethod was followed. Briefly, cells were washedwith 1× PBS following treatment and fixed in 10%paraformaldehyde for 1 h, permeabilized in 500 μL of1× cytoperm buffer for half an hour and blocked in3% BSA for 2 h. Immuno labeling was performed us-ing primary polyclonal rabbit antibody with dilution1:1000 and was incubated for 1 h, followed by stainingwith secondary polyclonal goat antirabbit antibody in

1:200 dilution conjugated to fluorescein isothiocyanate(FITC) fluorochrome and incubated in dark for halfan hour. The immuno-stained cells were then acquiredthrough flow cytometer (FACS Calibur, BD IS, San Jose,CA, USA). Cell-associated fluorescence in FL1 chan-nel was analyzed for 10,000 total events and in somecases the cells were double labeled with phycoerythrin-conjugated antibody (anti-CD-45 mAb) to confirm thegating for lymphocytes.

Evaluation of Apoptosis

DNA Laddering Profile by Agarose Gel Elec-trophoresis

To determine internucleosomal DNA fragmenta-tion (ladder), cells following culture were isolated,washed twice in 1× PBS and resuspended in 200-μLPBS, and centrifuged at 3,000 rpm for 1 min. Each sam-ple was then treated with 200 μL of lysis buffer for10 min at 15–25◦C. Isopropanol of 100 μL was added tothe samples and were transferred to the spin columntubes and centrifuged at 8,000 rpm for 1 min. The sam-ples were then washed twice with 500 μL of washingbuffer. Finally, DNA was eluted in prewarmed elutionbuffer by the centrifugation at 8,000 rpm for 1 min. Thesamples were dissolved in a 15 μL of loading bufferand subjected to electrophoresis in a 1% agarose gel.

Annexin-V-FITC/PI Assay

Measurement of lymphocyte apoptotic index wasperformed using Annexin-V-FITC/PI apoptosis assayas per manufacturer’s recommendations. From eachcell, forward light scatter (FSC), orthogonal light scat-ter (SSC), Annexin-V-FITC, and PI fluorescence weremeasured using Cell-Quest Software (BD-IS, USA). Thegate was applied in the FSC/SSC dot plot to restrictthe analysis to lymphocytes only. For the gated cells,the percentage Annexin-V-FITC positive or negative orPI positive or negative cells were evaluated. In eachcase, a total of 10,000 events were recorded in HI modewith 10/10 log quadrant gate.

Mitochondrial Membrane Potential DetectionAssay

Mitochondrial membrane potential of the cells wasdetected by staining cells with JC-1 (5, 5′, 6, 6′-tetra-chloro-1, 1′, 3, 3′-tetraethylbenzimidazolcarbocyanineiodide). The assay was carried out as per supplier’sinstructions. The gate was applied in the FSC/SSC dotplot to restrict the analysis to lymphocytes only. For thegated cells, the ratio of FL1/FL2 was evaluated. In eachcase, a total of 10,000 events were recorded in HI mode.

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Evaluation of Oxidative Stress andAntioxidant Defense System

Assay of Intracellular ROS

A fresh stock solution of CM-H2DCFDA (5 mM)was prepared in DMSO and diluted to a final concen-tration of 1 μM in 1× PBS. The cells were washed with1× PBS followed by incubation with 50 μL of work-ing solution of fluorochrome marker CM-H2 DCFDA(final working concentration adjusted to 2.5 μg/50 μL)for 2 h. The cells were harvested, washed in PBS, andcell-associated fluorescence was measured by flow cy-tometry in FL1 channel.

Evaluation of Oxidative Stress by HT 8-oxo-dGELISA

ELISA for quantification of 8-oxo-dG in culturesupernatant was performed as per manufacturer’s in-struction, and optical density was measured at 450 nmon an ELISA reader (Tecan Sunrise, Austria).

ELISA for Estimation of GR Activity

Levels of antioxidant defense system enzyme, GRwas measured using instructions as supplied by themanufacturer and absorbance kinetics was measuredat 340 nm through an ELISA reader.

FIGURE 1. DNA damage response in cultured human lymphocytes treated with N-succinimidyl N-methylcarbamate. (a) Dose responsefollowing treatment with concentrations 0.1, 1, 2.5, 5, 10, 50, and 100 μg in recipient cells showing ATM and γH2AX phosphorylation states after6 h of incubation period; (b) time course of ATM and γH2AX phosphorylation states following treatment with 5 μg concentration.

Multiplex CBA Assay for HumanInflammatory Cytokines

Supernatants collected from lymphocyte cultureswere subjected for measuring inflammatory responseby determining levels of cytokines, IL-8, IL-1β, IL-6, IL-10, TNF, and IL-12p70, and the assay was performedas per the manufacturer’s instructions. Data acquisi-tion and analysis were carried out on a flow cytometricplatform using BD CBA software.

RESULTS

DNA Damage Response

ATM and γH2AX Phosphorylation

Percent ATM phosphorylation measured in cul-tured lymphocytes through flow cytometry rangedfrom 6.02% in 0.1 μg to 71.23% in 100 μg at 6 hafter treatment in comparison to control, that is, 3.02%(Figure 1a) and showed a dose-dependent response,whereas, in the time-course experiments, an increas-ing trend was observed till 1 h, reaching a maximum of33.98% (Figure 1b). A similar dose-dependent responsewas also observed in phosphorylated states of γH2AXranging from 4.66% in 0.1 μg to 71.96% in 100 μg(Figure 1a), whereas a peak activity in the time-courseexperiment at 3 h with 42.70% following treatment with5 μg concentration was observed (Figure 1b).

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FIGURE 2. Effect of N-succinimidyl N-methylcarbamate on DNAfragmentation of cultured human lymphocytes showed a dose-dependent increase following 6 h of incubation period. Lane 1: MWmarker; Lane 2: Control; Lane 3–6: treated with 5, 10, 50, 100 μg,respectively.

Evaluation of Apoptosis

Apoptotic DNA Ladder

The typical DNA ladder, a hallmark of apoptosisdetected by agarose gel electrophoresis, demonstrateddose-dependent increasing pattern of DNA fragmen-tation in lymphocytes treated with N-succinimidylN-methylcarbamate following 6 h of incubation period(Figure 2).

Annexin-V-FITC/PI Assay

Annexin V is a Ca2+-dependent phospholipid-binding protein with high affinity for phosphatidyl ser-ine (PS). This protein is used as a sensitive probe for PSexposure upon the outer leaflet of the cell membraneand suitable for detection of apoptotic cells. Numberof apoptotic cells was incremental with increase inN-succinimidyl N-methylcarbamate dose and time pe-riod (Figure 3). At 100 μg concentration the apoptoticindex was maximum at 69.59% compared to control,that is, 2.6% (Figure 4a), whereas, in the time-coursestudy, a peak percentage of cells undergoing apoptosis(71.26%) was observed following 21 h after treatment(Figure 4b).

Mitochondrial Membrane Potential DetectionAssay

Membrane-permeable lipophilic cationic fluo-rochrome JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolcarbocyanine iodide) is used asa probe of transmembrane potential (�ψ). JC-1 pen-etrates into cells and its fluorescence is a reflection of�ψ . This assay showed a trend similar to that observedin Annexin-V/PI assay (Figure 5), which was dose andtime dependent. Maximum cells with depolarized mi-tochondria (loss of �ψ) was found at 100 μg concen-tration, that is, 60.89% (Figure 4a), whereas, in timecourse study, the percentage of cells showing depolar-ized mitochondria was observed at 21 h after treatment(Figure 4b).

Evaluation of Oxidative Stress andAntioxidant Defense System

Intracellular ROS Generation

The production of intracellular ROS was measuredby DCFH oxidation. The CM-H2DCFDA reagent pas-sively diffuses into cells wherein it is hydrolyzed by in-tracellular esterase to liberate 2′-7′-dichlorofluoressein,which, during the reaction with oxidizing species,yields a highly fluorescent compound 2′-7′- dichloroflu-orescein (DCF) that is trapped inside the cell [21]. Incells treated with N-succinimidyl N-methylcarbamatewith a fixed dose (5 μg) during different time course,there was a consistent increase, which appeared earlyand persisted for at least 6 h following treatment. Incontrast, a near twofold increase in H2O2 levels wasobserved at 12 h (Figure 6). At 5 μg concentration, a six-fold increase was observed in comparison to the control(Table 1).

HT 8-oxo-dG ELISA

8-Oxo-dG is a modified nucleoside base, which isthe most commonly studied and detected byproductof DNA damage that is excreted upon DNA repair.An increase in oxidative stress was observed in dose-dependent and time-course experiments with maxi-mum concentrations of 156.24 and 154.24 ng/mL ofaccumulated 8-oxo-dG at 100 μg concentrations and24 h, respectively (Table 1).

ELISA for Estimation of GR Activity

Glutathione reductase plays an essential role inmaintaining the appropriate levels of intracellular re-duced GSH [18]. GSH, long known for its protective

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FIGURE 3. Flow cytometric analysis for the apoptosis in cultured human lymphocytes following treatment with N-succinimidylN-methylcarbamate. (a) FSC/SSC plot showing the population of lymphocytes; (b) control cells; (c–f) cells treated with 5, 10, 50, 100 μgshowing dose-dependent increase in apoptotic index following 6 h of incubation period. Apoptotic index is the sum of the percentage of cellsthat are positive for Annexin-V-FITC alone and cells positive for both Annexin-V-FITC and propidium iodide (PI) within a population of cells.

function against oxidative cell damage [22], is thoughtto play a regulatory role in various lymphocyte func-tions. In our study, the maximum inhibition in the GRactivity was observed at 100 μg concentration and 24 h,respectively (Table 1).

Multiplex CBA Assay for HumanInflammatory Cytokines

Cytokines secreted by cells of the immune systemcan alter the behavior and properties of immune orother cells. At a site of inflammation, sets of cytokines

interact with immune cells, and their combined effectis often more important than the function of one iso-lated component [23]. Multiplex CBA assay for humaninflammatory cytokines displayed a dose-and time-dependent increase in response of inflammatory cy-tokines, IL-8, IL-1β, IL-6, IL-10, TNF, and IL-12p70in lymphocyte culture supernatant treated withN-succinimidyl N-methylcarbamate. Representativedata for dose-and time-dependent increase in levels ofcytokines secreted in culture supernatant evaluated bymultiplex CBA assay following 24 h of treatment with 5μg concentration and at 6 h after 100 μg dose treatmenthas been shown (Table 2).

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FIGURE 4. Exposure of N-succinimidyl N-methylcarbamate induces apoptosis in human lymphocytes. (a) Percentage of cells showingAnnexin-V binding and mitochondrial depolarization following treatment with N-succinimidyl N-methylcarbamate at concentrations rang-ing from 0.1 to 100 μg in recipient cells after 6 h of incubation period; (b) time course of Annexin-V binding and mitochondrial depolarizationin lymphocytes treated with 5 μg of N-succinimidyl N-methylcarbamate.

DISCUSSION

Isocyanates are considered as highly reactivemolecules because of their potential to modifybiomolecules under physiological conditions. Thesecompounds form covalent adducts with critical macro-molecules such as nucleic acids, resulting in a series ofbiotransformation events that initiate with the genera-tion of the reactive intermediates [24–27]. DNA damageleading to the cellular demise in mammalian cells upontreatment with isocyanates has been reported [28]. Ithas also been shown that “carbamate,” the reactive in-termediate of isocyanates, also induces the analogousupshot [29]. MIC, one of the most toxic isocyanates, isknown to exert immunological, mutagenic, and geno-toxic alterations [30–32], and because MIC is an im-portant byproduct of diverse industrial applications,we evaluated the immunotoxicity of MIC on culturedhuman lymphocytes isolated from healthy individu-als. DNA damage response, apoptosis, oxidative stress,and status of secreted inflammatory cytokines in the re-cipient cells were evaluated using N-succinimidyl N-methylcarbamate, a MIC substitute [20].

In response to double-strand breaks, the cell trig-gers checkpoints that halt the cell cycle. whereas adecision is made regarding repair and survival ordeath [33]. Only a few strand breaks are sufficientenough to initiate ATM response and activation of ATMthrough its autophosphorylation (Ser1981) results inrecruitment of many downstream signaling proteins

that modulate numerous damage response pathways[34]. This activation of ATM results in the subsequentphosphorylation of H2AX at Ser139 (γH2AX). Ourexperiments on cultured human lymphocytes showedphosphorylation of ATM and γH2AX that increased inexponential fashion in both dose-and time-dependentmanner (Figures 1a and 1b) indicative of DSBs forma-tion and damaged sites of the nuclear content upontreatment with N-succinimidyl N-methylcarbamate incomparison to controls.

Previous studies conducted on isocyanatederivative, such as isothiocyanates, suggest thatthey are capable to induce apoptosis [35]. Althoughthe exact mechanism of induction is still unknown,majority of studies have reported the involvementof mitochondrial-mediated pathway, that is, releaseof cytochrome c into the cytoplasm and activation ofcaspase 9 and caspase 3 [36–38]. The most prominentfeatures of apoptosis are DNA fragmentation andphosphatidylserine externalization. The present studyherein displayed an increased pattern of DNA frag-mentation in the form of apoptotic ladder at variousconcentration gradients posttreatment. Externalizationof PS and depolarization of the mitochondrial mem-brane of the recipient cells substantiated the formerand indicated a possible mitochondrial-mediatedevent.

The depolarized and uncoupled mitochondria mayincrease the generation of ROS. ROS are postulatedto induce cell death by promoting the leakage of

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FIGURE 5. Flow cytometric analysis for the mitochondrial depolarization in cultured human lymphocytes following treatment withN-succinimidyl N-methylcarbamate. (a) FSC/SSC plot showing the population of lymphocytes; (b) control cells; (c–f) cells treated with 5,10, 50, 100 μg showing dose-dependent increase in percent cells with depolarized mitochondria following 6 h of incubation period. Percentmitochondrial depolarization in cells that are positive for FL1-H (R3 zone) within a population of cells.

proapoptotic agents such as cytochrome c from the mi-tochondria via the opening of the permeability transi-tion pore, or by damage to the inner membrane. In ourstudy, a time- and dose-dependent increase in DCF flu-orescence indicative of H2O2 production was observedin the treated lymphocytes. In addition, increases inthe ROS generation also suggest an altered antioxidantdefense mechanism. Therefore, we, in our study, alsoevaluated the antioxidant defense states by the quan-titation of the activity of an enzyme that is GR. GRplays a vital role in defense against the toxicity of su-peroxide radical, and it has been well documented inprevious studies that any depletion in its activity can

cause deleterious effects on the cell [39]. Upon toxicexposure to MIC and its metabolites, GR is suscepti-ble to inhibition due to loss of its enzymatic activity[40,41]. Concordantly, GR activity was found to be in-hibited in lymphocytes exposed to N-succinimidyl N-methylcarbamate in a dose- and time-dependent man-ner, thereby, diminishing the antioxidant capacity ofthe cells. The maximum inhibitory effect was seen at100 μg concentration and 24 h , respectively.

An imbalance between oxidants and antioxidantsleads to the oxidative stress, which has been proposedto play an important role in the pathogenesis of manydiseases and elevated levels of DNA damage [42,43]. A

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FIGURE 6. Flow cytometric evaluation for the induction of ROS in cultured human lymphocytes labeled with CM-H2DCFDA. (a) FSC/SSCplot showing the population of lymphocytes; (b) Control cells; (c–f) Cells treated with a fixed dose (5 μg) of N-succinimidyl N-methylcarbamateat 0, 3, 6, 12 h showing time-dependent increase in percent induction of ROS within a population of cells (M2 zone).

biomarker of oxidative stress is 8-oxo-dG, a modifiednucleoside base, which is detected as a byproduct ofDNA damage that is excreted upon DNA repair [44].The present study demonstrated an increase in 8-oxo-dG levels in both dose dependent- and time-course ex-periments.

Isocyanate derivatives (carbamates) are alsoknown to enhance the levels of inflammatory cytokinessuch as IL-6, TNF-α, and IL-1 β [45], whereas it isalso evident that acute and chronic inflammatory sta-tus acts as a mediator in various pathological disordersalthough the exact mechanisms are unknown [46]. Thecapacity of inflammatory cells to generate and release

a spectrum of ROS and free radicals during an oxida-tive burst and their probable role in tumor productionare well documented [47–50]. In the present study, sta-tus of inflammatory cytokines (IL-8, IL-1β, IL-6, IL-10,TNF, and IL-12p-70) estimated in culture supernatantof treated lymphocytes through multiplex cytometricbead array assay following treatment showed a dose-dependent response.

In conclusion, the findings of the present investiga-tion provide insights to the understanding of immuno-toxic effects of methyl isocyanate at the genomic level.Precisely, methyl isocyanate induces DNA damage, in-creased intracellular ROS generation due to oxidative

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TABLE 1. Effect of N-Succinimidyl N-Methylcarbamate on Induction of Oxidative Stress (Formation of 8-Hydroxy-2′deoxyguanosine), Depletion of Antioxidant Defense States (GR) and Generation of H2O2 (CM-H2DCFDA) in Cultured HumanLymphocytes: Dose Response and Time Course

Dose Response

Concentration (μg) 8-oxo-dG (ng/mL) GR (mU/mL) CM-H2DCFDA (%)

Control 106.5 ± 16.17 471.4 ± 26.89 40.92 ± 2.945 128.94 ± 19.92 330 ± 28.5 59.73 ± 1.2910 131.86 ± 13.82 289.7 ± 26.69 59.45 ± 2.2350 133.41 ± 17.25 271.4 ± 23.58 67.13 ± 0.87100 156.24 ± 11.05 228.5 ± 14.2 40.92 ± 2.94

Time Course

Time (h) 8-oxodG (ng/mL) GR (mU/mL) CM-H2DCFDA (%)

Control 91.77 ± 18.58 479.5 ± 33.16 1.097 ± 0.103 111.3 ± 10.23 352 ± 11.95 34.45 ± 3.546 129.47 ± 28.07 324 ± 26.81 42.71 ± 3.3012 136.16 ± 16.55 267.8 ± 27.97 57.04 ± 5.3724 154.24 ± 17.84 194.23 ± 26.95 44.75 ± 2.49

TABLE 2. Inflammatory Cytokine Response in Cultured Human Lymphocytes Following Treatment with N-Succinimidyl N-Methylcarbamate. Representative Data for Dose- and Time- Dependent Increase in Levels of Cytokines Secreted in CultureSupernatant Evaluated by Multiplex CBA Assay Following 24 h of Treatment with 5 μg Concentration and at 6 h after 100 μgDose Treatment Has Been Shown

Cytokine Levels (pg/mL)

Treatment IL-8 (pg/mL) IFN-γ (pg/mL) TNF (pg/mL) IL – 1β (pg/mL) IL – 6 (pg/mL) IL-12p70 (pg/mL)

Control 111.4 ± 19.27 123.1 ± 20.09 140.7 ± 21.69 69.9 ± 19.5 51.3 ± 6.03 39.8 ± 3.9Following 24 h of treatment with 5 μg 231.9 ± 11.93 265.9 ± 25.8 218.5 ± 20.1 97.9 ± 14.9 83.1 ± 25.2 70.3 ± 17.3concentrationFollowing 6 h of treatment with 100 μg 317.6 ± 27.59 333.4 ± 32.4 301.7 ± 27.8 134.1 ± 13.2 113.9 ± 18.2 119.8 ± 14.0concentration

burst, with altered antioxidant defense mechanism, el-evated proinflammatory cytokine response via activa-tion of a mitochondrial mediated pathway, finally lead-ing to an inexorable cellular demise. We anticipate thatthese data along with other studies reported in the lit-erature would help to design better approaches in riskassessment of occupational and accidental exposure toisocyanates.

ACKNOWLEDGMENTS

The authors are thankful to the Bhopal Memo-rial Hospital Trust, India for financial support andMr. Naveen Kumar Khare for providing the necessarytechnical assistance.

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