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. Author manuscript; available in PMC: 2024 Jul 15.
Published in final edited form as: Toxicol Lett. 2019 Dec 26;322:1–11. doi: 10.1016/j.toxlet.2019.12.023

Toxic consequences and oxidative protein carbonylation from chloropicrin exposure in human corneal epithelial cells

Dinesh G Goswami a,1, Rama Kant a, David A Ammar a,2, Chapla Agarwal a, Joe Gomez a, Rajesh Agarwal a, Laura M Saba a, Kristofer S Fritz a, Neera Tewari-Singh a,b,*
PMCID: PMC11249040  NIHMSID: NIHMS1985155  PMID: 31884112

Abstract

Chloropicrin (CP), a warfare agent now majorly used as a soil pesticide, is a strong irritating and lacrimating compound with devastating toxic effects. To elucidate the mechanism of its ocular toxicity, toxic effects of CP (0–100 μM) were studied in primary human corneal epithelial (HCE) cells. CP exposure resulted in reduced HCE cell viability and increased apoptotic cell death with an up-regulation of cleaved caspase-3 and poly ADP ribose polymerase indicating their contribution in CP-induced apoptotic cell death. Following CP exposure, cells exhibited increased expression of heme oxygenase-1, and phosphorylation of H2A.X and p53 as well as 4-hydroxynonenal adduct formation, suggesting oxidative stress, DNA damage and lipid peroxidation. CP also caused increases in mitogen activated protein kinase-c-Jun N-terminal kinase and inflammatory mediator cyclooxygenase-2. Proteomic analysis revealed an increase in the carbonylation of 179 proteins and enrichment of pathways (including proteasome pathway and catabolic process) in HCE cells following CP exposure. CP-induced oxidative stress and lipid peroxidation can enhance protein carbonylation, prompting alterations in corneal epithelial proteins as well as perturbing signaling pathways resulting in toxic effects. Pathways and major processes identified following CP exposure could be lead-hit targets for further biochemical and molecular characterization as well as therapeutic intervention.

Keywords: Chloropicrin, Pesticide, Corneal injury, Oxidative stress, Protein carbonylation

Graphical Abstract

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1. Introduction

Chloropicrin (CP, PS, CCl3NO2, nitrochloroform, Trichloronitromethane), an aliphatic nitrate compound, was first discovered in 1848 and was employed during World War I as a warfare agent. It was used within a mixture with other toxic gases for its toxicity, and irritating, choking as well as powerful lacrimating tear gas like properties (AEGL, 2008; Sutherland, 2008). It is an aliphatic colorless liquid and is currently used as a broad spectrum fumigant and pesticide in agriculture (AEGL, 2008; Ruzo, 2006). Apart from its toxicity to insects and nematodes, CP exposure is harmful to humans and other mammals, affecting all body surfaces (AEGL, 2008). Decomposition of CP can release toxic reactive gases such as chlorine, phosgene, and oxides of nitrogen (Huebner and Trickey, 2013). There are potential health risks from soil fumigation with CP, in addition to the danger of accidental CP exposures; a total of 1015 cases (from 1992 to 2007) were reported to the California Pesticide Illness Surveillance Program (Barry et al., 2010). Due to its volatile nature, eyes, skin, and the respiratory system are the main target tissues after CP exposure and are also the most severely affected. There have been reports of dry cough, sinus irritation, and inflammation of the oropharynx in addition to excessive lacrimation with eye pain, vertigo, fatigue and headache. In addition to its accidental or occupational exposure, toxic effects of CP, together with its easy availability and lack of antidotes make it a potential agent for warfare and terrorism (AEGL, 2008; Oriel et al., 2009).

Though CP has extremely toxic and irritating effects on the eyes, skin, and respiratory system, research efforts to evaluate the pathogenesis and mechanisms of injuries, mainly to the maximally affected ocular tissue are elusive (Pesonen et al., 2014). CP exposure-induced eye injury manifests as eye irritation, associated with lacrimation and inflammation, which involves corneal edema, damage to ocular tissues, and could ultimately lead to visual damage (AEGL, 2008; O’Malley et al., 2004). However, specific biomarkers to assess ocular exposure are not available, and a critical gap exists in our knowledge regarding the pathogenesis of CP-induced eye injury. These limitations have thus hampered the development of interventions for ocular injury management that can be used to reduce and/or treat toxicity due to CP exposure.

Limited published reports on CP exposure in the lung, bronchiolar and airway epithelial cells, and cells of the retinal pigmented epithelium have suggested a role of oxidative stress, p53 accumulation, and activation of mitogen activated protein kinases (MAPKs) in CP induced toxicity (Pesonen et al., 2017, 2015, 2014, 2012). However, comprehensive studies on the mechanism of CP toxicity upon ocular exposure are elusive. CP induced oxidative stress could lead to the generation and accumulation of reactive oxygen species (ROS). ROS can further modify lipids, proteins, and DNA. Oxidative modification of lipids and accumulation of reactive aldehydes such as 4-hydroxynonenal (4-HNE) and subsequent protein carbonylation could be a major contributors of protein modification leading to cellular toxicity and ocular tissue damage (Barrera, 2012). Lipid peroxidation and generation of 4-HNE is reported to be involved in the activation of oxidative stress-related pathways and transcription factors (Barrera, 2012). The detrimental consequences from altered protein function due to lipid peroxidation are related to disruptions in cellular signaling and could play an important role in the progression of toxicity as observed in numerous diseases related to oxidative stress, which could also arise from CP exposure (Halliwell, 2000; Pesonen et al., 2017, 2015, 2012; Ramana et al., 2017; Shichiri, 2014).

Cornea, the outermost layer of the eye and most densely innervated, is maximally exposed and is highly sensitive to chemical and toxic environmental exposures; however, detailed studies on the effect of CP in the corneal cells are missing. Hence, to study the toxic consequences and the related mechanisms of CP exposure in the corneal tissue, human corneal epithelial (HCE) cells were employed. In this study, we have elucidated the cytotoxic consequences of CP exposure and examined the molecular mechanisms including protein carbonylation, which could be important contributors of CP-induced mechanistic alterations and corneal injury.

2. Materials and methods

Chemicals and Reagents.

CP, N-Acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), antibeta-actin antibody, hoechst 33342 and all other chemicals were from Sigma-Aldrich (St. Louis, MO). Keratinocyte-SFM media, trypsin-EDTA, and 100 × antibiotic-antimycotic solutions were from Thermo-Fisher Scientific (Waltham, MA). Primary antibodies for phosphorylated Junamino-terminal kinase (JNK1/2; Thr183/ Tyr185), JNK1/2, phosphorylated p38 (Thr180/Tyr182), p38, phosphorylated p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), phosphorylated H2A.X (Ser139), phosphorylated p53 (Ser15), p53, heme oxygenase-1 (HO-1), cleaved-poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, anti-mouse and anti-rabbit IgG HRP−conjugated secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti 4-HNE antibody was purchased from Alpha-Diagnostic (San Antonio, TX). The detergent compatible (DC) protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). Enhanced chemiluminescence kit (ECL) was from GE healthcare Bio-Sciences (Pittsburgh, PA). Anti-cyclooxygenase-2 (COX-2) monoclonal antibody was from Cayman Chemical (Ann Arbor, MI). Goat anti-mouse IgG and anti-rabbit IgG secondary antibody labeled with either IRDye 680 LT or IRDye 800CW were purchased from LI−COR Biosciences (Lincoln, NE). EZ-link biotin hydrazide was purchased from Thermo-Fisher Scientific (Waltham, MA) and anti-biotin HRP antibody was from GeneTex Inc. (Irvine, CA).

2.1. Cell culture and CP exposure

HCE cells were purchased from Thermo-Fisher Scientific (Waltham, MA) and were cultured per the vendor’s protocol using keratinocyte-SFM media supplemented with epidermal growth factor, bovine pituitary extract, and 1 % antibiotic-antimycotic solution under standard cell culture conditions. When cells reached 60–70 % confluence, they were exposed to CP (0–100 μM) continuously for the entire length of the experiment (24 h). A 2 M stock of CP was prepared in DMSO, diluted in media, and added, within 10 min. of its preparation, to the cells to achieve desired concentrations. For NAC pre-treatment, cells were pretreated with 5 mM NAC for 1 h before CP exposure.

2.2. Cell viability assay and measurement of apoptotic cell death

Cells were seeded in 96-well culture plates, grown overnight under standard cell culture conditions and exposed to either media alone (control) or CP (0–100 μM). Following 24 h of CP exposure, cell viability was measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as reported earlier (Tewari-Singh et al., 2010). Hoechst staining for the quantification of apoptotic cells was performed as reported earlier (Tewari-Singh et al., 2010). Briefly, at 60–70 % confluence, the cells were exposed to either media alone (control) or CP for 24 h. After 24 h, cells were collected, washed with PBS and stained with Hoechst 33342 (1 mg/mL). Quantification of apoptotic cells was performed in triplicate, and 100 cells were counted per sample in different fields to score for live and apoptotic cells. Cells were observed and quantified using a fluorescence microscope (Nikon Eclipse TE300) equipped with Nikon DS-Fi2 camera using NIS elements BR software. The cells were classified as live cells (light blue fluorescence and normal structure) and apoptotic cells (bright blue florescence and enlarged with condensed or fragmented chromatin).

2.3. Western blot analyses

HCE cells were grown in 100 mm culture dishes and exposed to 50 μM CP, and lysates were prepared 24h after the exposure. Protein concentration was estimated using Bio-Rad DC protein assay kit (Hercules, CA) and western blot analyses for DNA damage and apoptotic cell death markers [H2A.X and p53 phosphorylation, p53 accumulation, cleaved caspase-3, and cleaved-PARP], MAPKs activation [JNK1/2, ERK1/2, and p38 phosphorylation and accumulation], inflammatory mediator [COX-2], and oxidative stress marker [HO-1] expression were carried out as described previously (Pal et al., 2009; Tewari-Singh et al., 2010). Each membrane was used for assessing multiple proteins and re-probed with anti-β-actin antibody as loading control. All detected bands were scanned using Adobe Photoshop 6.0 (Adobe Systems, Inc., San Jose, CA). Results obtained were quantified via densitometric analysis of bands using the Image J Program (NIH, Bethesda, MD).

2.4. 4-HNE and Protein carbonylation analyses

HCE cells were grown in 100 mm culture dishes and exposed to 50 μM CP, and lysates were prepared 24 h after the exposure. Protein concentration was estimated using Bio-Rad DC protein assay kit (Hercules, CA). Western blot analyses including that of 4-HNE was performed as reported earlier (Pal et al., 2009; Tewari-Singh et al., 2010). Hydrazide chemistry was employed to derivatize protein carbonyls from the protein samples. Briefly, 50 μg of the protein sample was incubated overnight in dark with 5 mM biotin hydrazide at room temperature. After the incubation, samples were denatured in loading buffer and resolved on a 12% Tris-glycine gel and were transferred to nitrocellulose membrane. The membrane was than blocked with 5% milk in PBS at room temperature for 1 h and was blotted for biotinylated proteins using anti-biotin antibody (1:5000 in 5% milk in PBS). The signal was detected by enhanced chemiluminescence detection.

2.5. Proteomics analyses of protein carbonylation

To identify the targets of protein carbonylation, whole cell lysates from CP-exposed cells were subjected to mass spectometry (MS) analysis using a hydrazide-dependent approach. The specific sites of protein carbonylation were determined using biotin-hydrazide enrichment and a nanoAdvanced UPLC (Bruker) with a 15cmx100um ProntoSil C18AQ column and 2 cm trap column (nanoLCMS Solutions). Mobile phase was H2O + 0.1%FA (A) and acetonitrile + 0.1%FA (B), peptides were separated using a gradient of 2–40 % B over 30 min at a flow rate of 800 nL/min with the column temperature of 40 °C. The column was connected through a Captive Spray nano source to an Impact Q-tof (Bruker). Data was processed using DataAnalysis 4.2 (Bruker) and compounds were searched against the SwissProt database using Mascot 2.4 (Matrix Science) with the percolator algorithm. Protein and peptide results were assessed and filtered with ProteinScape 3.0 (Bruker).

2.6. Bioinformatics analyses

To uncover biological pathways that may be impacted by protein carbonylation after CP exposure, we used a differential enrichment analysis. Initially, the set of proteins that were carbonylated in the CP exposure group and not in the control were examined for functional enrichment of Gene Ontology (GO) terms (Ashburner et al., 2000) using the statistical overrepresentation test in the PANTHER (protein annotation through evolutionary relationship) classification system (Version 11.0). (Mi et al., 2013) For KEGG pathway (Kanehisa et al., 2016) enrichment, the KEGG 2016 database was downloaded from Enrichr (Kuleshov et al., 2016) and a Fisher’s exact test was executed in R statistical software (version 3.5.1; http://www.r-project.org). To ensure that the enrichment observed for protein carbonylation data was not simply due to enrichment for carbonylated proteins in this particular cell type, we compared the functional enrichment of the CP only carbonylated proteins to the enrichment of carbonylated proteins detected in BOTH the control group and the CP exposed group using a differential enrichment analysis. Enriched GO terms/KEGG pathways are reported if they are significantly enriched in the CP only group compared to all human proteins (p < 0.05) and if they are differentially enriched compared to the group of proteins that were carbonylated in both the control group and the CP-treated group (p < 0.05). Fold enrichment is calculated as then number of observed carbonylated proteins observed only in the CP treated cells compared to the number expected by chance. Overlap of proteins between enriched KEGG pathways and GO terms was visualized using Cystoscape (version 3.6.1) (Shannon et al., 2003).

2.7. Statistical analyses

Statistically significant differences between groups were determined by one-way ANOVA using SigmaStat 3.5 software (Jandel Scientific, San Rafael, CA) and the Tukey test for multiple comparisons, and a p-value of < 0.05 was considered significant. Data are represented as mean ± SEM.

3. Results

3.1. CP exposure reduced cell viability and caused an increase in apoptotic cell death

Cellular toxicity is one of the key responses upon exposure to hazardous or environmental chemical agents. Therefore, we first analyzed the effect of CP exposure on cell viability of HCE cells using MTT assay. CP exposure resulted in a significant dose-dependent decrease in cell viability (Fig. 1A). The cell viability reduced to 77, 65, 39 and 22% upon exposure to 25, 50, 75 and 100 μM CP, respectively, in comparison to control untreated cells. Since the CP-induced cell death could be via signaling pathways and apoptotic in nature, we next analyzed the effect of CP on apoptotic cell death in HCE cells. Hoechst-staining of the CP-exposed HCE cells showed a significant (p > 0.05) 3-fold increase in apoptotic cell death upon exposure to 50 μM CP as observed by fluorescence microscopy (Fig. 1B). CP-induced cell death could be related to DNA damage, activation of p53 and PARP-caspase pathways. Hence, we next analyzed the effect of CP exposure on related molecules.

Fig. 1. CP exposure reduced cell viability and caused apoptotic cell death in HCE cells.

Fig. 1.

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HCE cells were exposed to CP (0–100 μM) for 24 h and subjected to MTT assay (A) for cell viability and Hoechst-staining for apoptotic cell death assay (B) as detailed under the Materials and Methods section. Data presented are mean ± SEM (n = 3–6); *, P < 0.05 as compared with control group; C, control; CP, chloropicrin.

3.2. CP exposure resulted in cytotoxicity that was accompanied with changes in DNA damage, inflammatory and oxidative stress markers in HCE cells

CP exposure led to severe DNA damage, as evident by a strong increase in the phosphorylation of H2A.X (Ser139), one of the markers for double stranded DNA breaks (Fig. 2A). Twenty-four hours after exposure to 50 μM CP, a 7-fold increase in the phosphorylation of H2A.X was observed. A 2-fold increase in the phosphorylation of p53 (Ser15), was also observed (Fig. 2A). The role of caspase-poly ADP ribose polymerase (PARP), one of the executors of apoptosis, was next analyzed. CP (50 μM) exposure to HCE cells resulted in a 32- and 9-fold increase, respectively, in cleaved caspase-3 and cleaved PARP levels in comparison to control untreated cells (Fig. 2A).

Fig. 2. CP exposure resulted in DNA damage and apoptotic cell death, which was accompanied with the activation of MAPKs, and alterations in inflammatory mediators and oxidative stress markers in HCE cells.

Fig. 2.

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HCE cells were exposed to 50 μM CP for 24 h. After exposure, cell lysates were prepared and western immunoblotting for DNA damage and apoptotic cell death markers [H2A.X and p53 phosphorylation, p53 accumulation, cleaved caspase-3, and cleaved-PARP (A)], MAPKs activation [JNK1/2, ERK1/2, and p38 phosphorylation and accumulation (B)], inflammatory mediator [COX-2 (C)], and oxidative stress marker [HO-1 (D)] expression was carried out as detailed under Materials and Methods section. Protein loading was checked by stripping and re-probing the membranes for beta-actin. The results obtained were quantified by densitometric analyses of the immunoblots and fold change compared to control was calculated. C, control; CP, chloropicrin.

Next, we investigated the involvement of MAP kinases JNK, p38, and ERK in CP induced cytotoxicity. CP exposure caused a 5-fold increase in JNK1/2 phosphorylation, with slight increase in total JNK1/2 levels (Fig. 2B). Increase in the phosphorylation of ERK and p38 following CP exposure was also observed (a 1.1- and 1.3-fold increase, respectively, in phosphorylation of ERK1/2 and p38); with decrease in total ERK and total p38 levels (Fig. 2B).

Levels of inflammatory mediator COX-2 in CP-induced injury was next evaluated as it is a key enzyme involved in prostaglandin biosynthesis and serves as a marker for inflammation. (Norregaard et al., 2015) A 3-fold increase in the expression of COX-2 was observed upon CP exposure (Fig. 2C). Next the levels of anti-oxidant, HO-1, a stress protein and oxidative stress marker was analyzed. Similar to COX-2, a 3-fold increase in HO-1 expression was observed at 24 h post- 50 μM CP exposure in HCE cells (Fig. 2D).

3.3. NAC pre-treatment ameliorated CP-induced changes in DNA damage, inflammatory and oxidative stress markers in HCE cells

Since CP exposure could involve oxidative stress, we next analyzed the effect of antioxidant, NAC on the toxic consequences of CP exposure in HCE cells. NAC pre-treatment led to a complete reversal in CP-induced double stranded DNA break marker H2A.X (Ser139) and p53 (Ser15) phosphorylation (Fig. 3A). NAC pre-treatment also mitigated CP-induced increase in cleaved PARP levels in the HCE cells (CP exposure resulted in 11.7-fold increase in cleaved PARP levels which was reduced to 1.6-fold in NAC pre-treated cells; Fig. 3A). In addition, NAC pre-treatment led to almost complete reversal in CP-induced increase in JNK1/2 phosphorylation (CP exposure resulted in 8.2-fold increase in pJNK1/2 levels which came down to baseline levels when cells were pre-treated with NAC before CP exposure; Fig. 3B). Similarly, CP-induced alteration in HO-1 level was also reversed markedly upon NAC pre-treatment (there was a 2.5-fold increase in HO-1 levels in CP exposed cells which was reduced to only 1.3-fold increase when cells were treated with NAC prior to CP exposure; Fig. 3C). Together, these results suggest that toxic response from of CP could be mitigated by anti-oxidant NAC-pretreatment.

Fig. 3. NAC pre-treatment ameliorated CP-induced changes in markers related to DNA damage, apoptotic cell death and oxidative stress, and MAPK phosphorylation.

Fig. 3.

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HCE cells. HCE cells were either exposed to CP (50μM) or pre-treated with NAC (5 mM NAC for 1 h) and then exposed to 50 μM CP for 24 h. After 24 h, cell lysates were prepared and western immunoblotting for H2A.X and p53 phosphorylation, p53 accumulation, and cleaved-PARP (A), JNK1/2 phosphorylation and accumulation (B), and HO-1 expression (C) was carried out as detailed under Materials and Methods section. Protein loading was checked by stripping and re-probing the membranes for beta-actin. The results obtained were quantified by densitometric analyses of the immunoblots and fold change compared to control was calculated. C, control; CP, chloropicrin; NAC, N-Acetyl-L-Cysteine.

3.4. CP exposure resulted in 4-HNE adducts formation and protein carbonylation in HCE cells

4-HNE, one of the biomarkers of lipid peroxidation, was next analyzed in HCE cells exposed to CP. Western blot analysis showed modification in two proteins at 24 h after exposure to 50 μM CP (Fig. 4A). Further confirmation of oxidative stress was carried out by analysis of protein carbonylation using biotin hydrazide labeling. Biotin hydrazide specifically reacts with the aldehyde groups on proteins and serves as a marker for proteins adducted by lipid oxidation products. A 24 h CP exposure (50 μM) resulted in a ~8-fold increase in protein carbonylation (Fig. 4B).

Fig. 4. CP exposure caused lipid peroxidation and protein carbonylation in HCE cells.

Fig. 4.

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HCE cells were exposed to 50 μM CP for 24 h. After exposure, cell lysates were prepared and western immunoblotting for 4-HNE (A) and protein carbonylation (B) for 24 h exposure was carried out by western blotting as detailed under Materials and Methods section. Protein loading was checked by stripping and re-probing the membrane for beta-actin and the results were quantified by densitometric analysis and fold change compared to control was calculated. C, control; CP, chloropicrin.

3.5. Proteomic and bioinformatic analyses of carbonylated proteins in HCE cells upon CP exposure

Mass-spec analyses using hydrazide chemistry revealed an increase in protein carbonylation upon CP exposure. A total of 347 unique carbonylated proteins were detected using this approach (Fig. 5A). Of the 347 carbonylated proteins identified, 142 were found in both control and CP exposed groups with 26 exclusively present in control and 179 found solely in CP-exposed HCE cells (Fig. 5A). To further elucidate the effect of protein carbonylation upon CP exposure in HCE cells, bioinformatics analyses were carried out. Functional enrichment analyses of the identified carbonylated proteins and their co-occurrence in the pathways found enrichment of the proteasome pathway (8-fold enrichment; differential enrichment p-value = 0.038), catabolic processes (2.4-fold enrichment; differential enrichment p-value = 0.041), intracellular protein transport (2-fold enrichment; differential enrichment p-value = 0.017), and tRNA aminoacylation for protein translation (18-fold enrichment; differential enrichment p-value = 0.016) as the major affected processes and pathways in HCE cells upon CP exposure (Fig. 5B; Table 1). In addition, carbonylated proteins identified in cells after CP exposure, but not in the control-retreated cells, were also enriched for localization in the mitochondrial inner membrane (16-fold enrichment; differential enrichment p-value = 0.031) and for localization in the protein complex (2-fold enrichment; differential enrichment p-value = 0.006).

Fig. 5. CP exposure caused increase in carbonylated proteins and altered various pathways and processes in HCE cells.

Fig. 5.

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HCE cells were exposed to 50 μM CP for 24 h. After 24 h, cell lysates were prepared. Proteomic analysis was carried out to identify carbonylated proteins via biotin-hydrazide enrichment and nUltra Performance Liquid Chromatography (nUPLC)-LC–MS/MS and further bioinformatics analyses of the obtained data was carried out as detailed under Materials and Methods section. Venn diagram showing the comparison of carbonylated proteins found post 24 h exposure in control and CP exposed HCE cells (A). Functional enrichment analysis, based on the KEGG pathway and gene ontology term assignments of the proteins identified and their co-occurrence in pathways, demonstrates critical pathways impacted by protein carbonylation in HCE cells upon CP exposure (B). C, control; CP, chloropicrin.

Table 1.

List of pathways, biological processes and cellular components altered significantly by CP exposure in HCE cells.

Category Number of Proteins In Background Observed Number of Proteins Only Identified in CP50 Proteins Only Identified in CP50 Expected Number of Proteins Only Identified in CP50 Fold Enrichment (CP50 only) P-value (CP50 only) Observed Number of Proteins Identified in CP50 and Control Proteins Identified in CP50 and Control Expected Number of Proteins Identified in CP50 and Control Fold Enrichment (CP50 and Control) P-value (CP50 and Control) Differential Enrichment P-value
KEGG PATHWAYS
Proteasome (hsa03050) 44 6 PSB3_HUMAN; PSMD_6HUMAN; PRS6B_HUMAN; PSD13_HUMAN; PRS4_HUMAN; PSMD2_HUMAN 0.75 8.0 0.0002 0 0.7 0.0 1.00 0.038
GO BIOLOGICAL PROCESS
catabolic process (GO:0009056) 789 16 AT2A3_HUMAN; DDB1_HUMAN; DDX21_HUMAN; DHX32_HUMAN; DX39B_HUMAN; EFTU_HUMAN; ETFA_HUMAN; GLSK_HUMAN; GNAI2_HUMAN; HXK1_HUMAN; IF2G_HUMAN; PRS4_HUMAN; PRS6B_HUMAN; PSD13_HUMAN; PSMD2_HUMAN; PSMD6_HUMAN 6.7 2.4 1.2E-03 5 ACADM_HUMAN; AT1A3_HUMAN; CATB_HUMAN; CATD_HUMAN; EF1A1_HUMAN 6.1 0.8 0.421 0.041
intracellular protein transport (GO:0006886) 1025 18 CALX_HUMAN; CAV1_HUMAN; CDC42_HUMAN; CLH1_HUMAN; COX2_HUMAN; CPNE3_HUMAN; LAMP1_HUMAN; LAMP2_HUMAN; QCR2_HUMAN; RBBP4_HUMAN; RHOA_HUMAN; RRAS_HUMAN; RRAS2_HUMAN; TBA1B_HUMAN; TBB4A_HUMAN; TIM50_HUMAN; TMEDA_HUMAN; TOM40_HUMAN 8.7 2.1 2.8E-03 5 ACTB_HUMAN; ACTC_HUMAN; ARL8A_HUMAN; HNRPK_HUMAN; RAP1B_HUMAN 8.0 0.6 0.187 0.017
tRNA aminoacylation for protein translation (GO:0006418) 47 7 ECHA_HUMAN; HCDH_HUMAN; RM39_HUMAN; SYDC_HUMAN; SYEP_HUMAN; SYQ_HUMAN; SYRC_HUMAN 0.4 17.7 2.0E-07 0 0.4 0.0 0.694 0.016
GO CELLULAR COMPONENT
mitochondrial inner membrane (GO:0005743) 46 6 COX5A_HUMAN; NDUS1_HUMAN; QCR1_HUMAN; QCR2_HUMAN; TIM50_HUMAN; TTC19_HUMAN 0.4 15.5 3.2E-06 0 0.4 0.0 0.699 0.031
protein complex (GO:0043234) 1154 23 CLH1_HUMAN; COX5A_HUMAN; DDB1_HUMAN; GBB2_HUMAN; GNAI2_HUMAN; METK2_HUMAN; NDUS1_HUMAN; NDUV2_HUMAN; OCAD2_HUMAN; PRP19_HUMAN; PRS4_HUMAN; PRS6B_HUMAN; PSD13_HUMAN; PSMD2_HUMAN; QCR1_HUMAN; RPAB3_HUMAN; TBA1B_HUMAN; TBB4A_HUMAN; TIM50_HUMAN; TMEDA_HUMAN; TOM40_HUMAN; VATA_HUMAN; VATG1_HUMAN 9.7 2.4 1.2E-04 6 ATPA_HUMAN; ATPB_HUMAN; PLAK_HUMAN; RPN1_HUMAN; RPN2_HUMAN; SMD1_HUMAN 9.0 0.7 0.202 0.006
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4. Discussion

CP, extensively used as a soil fumigant and pesticide, is a highly toxic and irritating agent and can cause severe ocular and respiratory damage (AEGL, 2008). It poses a threat to be used in warfare and in terrorist activities apart from its accidental and occupational exposure (Pesonen et al., 2014, 2010). Limited reports show that accidental or occupational exposure of CP to humans is related with nausea, vomiting, difficulty in breathing, nephritis, skin inflammation and lacrimation (Barry et al., 2010; Oriel et al., 2009). Although CP can cause serious damage to the most sensitive ocular tissue upon exposure, its mechanism of action is poorly understood, and effective antidotes are not available. In the present study, we demonstrate that oxidative stress-induced lipid peroxidation leading to subsequent molecular alterations and protein carbonylation could play a major role in corneal toxicity and tissue injury from CP exposure. Our study for the first time provides molecular insight into the corneal injury from CP exposure and could be helpful towards future screening and development of antidotes.

DNA damage, apart from a byproduct of metabolism, could be triggered by radiation and toxic chemicals (Ciccia and Elledge, 2010; Errol et al., 2006; Jackson and Bartek, 2009). H2A.X, member of histone H2A family, gets phosphorylated at Ser139 upon exposure to DNA damaging insults resulting in double strand breaks, and is a strong marker of DNA damage (Rogakou et al., 1998; Sedelnikova et al., 2002). p53 phosphorylation at Ser15 as well as total p53 level goes up following DNA damage that results in cell cycle arrest for DNA repair, and could lead to eventual cell death (Al Rashid et al., 2005; Jackson and Bartek, 2009; Lavin and Gueven, 2006). Our study outcomes showing CP-induced increases in the H2A.X and p53 phosphorylation as well as increase in p53 levels, suggests the role of H2A.X and p53 in CP-induced DNA damage and HCE cell death. Also, CP exposure in HCE cells led to activation of caspase-PARP pathways, triggering apoptotic cell death. Our results corroborate with earlier reports where CP exposure in human retinal pigmented epithelial (HRPE) cells and human lung epithelial (HLE) cells, has been shown to result in reduced cell viability and p53 accumulation (Pesonen et al., 2014, 2012). Along with DNA damage, oxidative stress could activate signaling pathways leading to the induction of inflammatory mediators (Reuter et al., 2010). The generation of 4-HNE adducts and protein carbonylation indicate that CP-induced oxidative stress could result in lipid peroxidation and activation of associated downstream signaling pathways. The generation of 4-HNE could be involved in the regulation of stress-related transcription factors such as multifunctional regulator nuclear factor erythroid 2-related factor (Nrf-2), activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) via activation of signaling pathways such as MAPK (Ayala et al., 2014). These alterations could lead to the CP-induced toxic responses including cell proliferation, and cell death (by apoptosis and necrosis).

The toxicity of CP could also be due to chlorine, released from CP under physiological conditions and formation of nitromethane by dehalogenation of CP (AEGL, 2008). Chlorine, upon reacting with cellular water could generate free radicals and hypochlorous acids resulting in the formation of reactive intermediates (Castro et al., 1983; Evans, 2005; Sparks et al., 1997; Yadav et al., 2010). CP exposure has been shown to perturb the redox state of the cell with depletion of major cellular anti-oxidant glutathione (Pesonen et al., 2012). It has also been reported to induce endoplasmic reticulum (ER) stress, leading to increase ROS production, and HO-1 expression in human HRPE and HLE cells (Pesonen et al., 2014, 2012). HO-1 is upregulated by various physiological and exogenous stress stimuli (heme, heavy metals, oxidative stress, inflammatory cytokines, heat stress and UV irradiation). HO-1 and its products function as adaptive molecules and play an important role in various biological responses, including inflammation, oxidative stress, cell survival and cell proliferation (Applegate et al., 1991; Poss and Tonegawa, 1997; Ryter and Choi, 2005; Stocker, 1990). HO-1 is reported to exhibit cytoprotective, anti-oxidant and anti-inflammatory properties which could be due to 4-HNE formation, Nrf-2 dependent as well as linked to ERK1/2 and transcription factor PI3K/Akt activation (Ayala et al., 2014; Feng et al., 2017). Hence, it will be important to further study the role of Nrf2-Keap1 (novel Nrf2-binding protein) signaling pathway on CP-induced oxidative stress and toxicity.

MAPKs are important signaling molecules and play a key role in regulating apoptosis, cell proliferation and differentiation, and various cellular events. MAPKs can activate AP-1, which plays a central role in activating various inflammatory, proteolytic and angiogenic mediators. MAPK-JNK found upregulated upon CP exposure is known to be a key molecule in modulating apoptosis signaling in response to oxidative stress (Boutros et al., 2008; Darling and Cook, 2014; Kim and Choi, 2010). Involvement of MAPKs (ERK1/2) has been shown in CP-induced lung injury, (Pesonen et al., 2014, 2012) and will be of further interest to see how these pathways regulate CP-induced signaling and oxidative stress.

NAC, a thiol and mucolytic agent and precursor of cysteine, pretreatment has been shown to be beneficial in treating CP-induced toxic consequences in HLE cells (Pesonen et al., 2014). Our results also show that NAC-pretreatment of HCE cells before CP exposure reduces DNA damage and caspase-PARP pathway markers. CP-induced activation of MAPK and HO-1 expression was also inhibited. HO-1 induction could also be related to oxidative injury and can serve as a marker for oxidative injury. Pre-treatment with NAC could decrease the oxidative injury and thus the HO-1 induction. NAC is a powerful scavenger of ROS/NOS and it also stimulates GSH synthesis, thus it further suggests the role of ROS in CP-induced toxicity.

Protein carbonylation is shown to play a role in a variety of diseases including cancer, neurological disorders, liver disease, inflammation and cardiovascular disease, associated with oxidative stress (Dalle-Donne et al., 2003; Fritz and Petersen, 2011). The reactive carbonyl compounds, mainly resulting from lipid peroxidation, accumulate during the oxidative stress-related diseases, which lead to carbonyl stress, damage to tissues, and pathological consequences including inflammatory response, apoptosis and cellular dysfunction (Fritz et al., 2011). One of the major limitations in evaluating these protein carbonyls is their potential instability during analysis and low abundance, which has been overcome through the years by utilizing enrichment strategies and mass spectrometric analyses (Suzuki et al., 2010). Utilizing modern strategies, similar to reported earlier (Coughlan et al., 2015), we observed an increase in the number of carbonylated proteins, following CP exposure in the cornea, in our study. To further identify the physiological impact of carbonylation we employed bioinformatics analyses of the identified carbonylated protein. One of the major cellular components affected upon CP exposure is the mitochondrial inner membrane complex, suggesting mitochondrial dysfunction, which could lead to reduced energy production. In fact, one of the major biological processes affected by CP-exposure is t-RNA amino acylation for protein translation, which is an energy consuming process. Earlier studies have suggested a rerouting of cellular energy production by CP to the less efficient glycolysis and pentose phosphate pathway (PPP) (Pesonen et al., 2017, 2015). This could be an attempt to restore the redox potential as PPP is the main pathway for generating NADPH. CP exposure has been shown to result in ER stress. ER serves important function of protein synthesis, modification, and transport. Some of the major processes found affected in our study upon CP exposure are protein assembly and protein transport, thus further confirming the role of ER stress in CP induced injury (Pesonen et al., 2012). Effect of CP on protein complex assembly further confirms the earlier reports of the involvement of UPR pathway (Pesonen et al., 2012) and warrants further investigation of this pathway in CP-induced corneal injury. Accumulation of misfolded proteins activates the proteasome to remove unwanted proteins. Importantly, we observed the proteasome pathway to be the major pathway affected upon CP-exposure (8-fold enrichment) in HCE cells. These results further confirm the earlier reports regarding similar pathways being affected as suggested by transcriptome analyses of CP-exposed lung epithelial cells (Pesonen et al., 2017, 2015).

Overall, our findings regarding CP-induce corneal toxicity contribute towards elucidating mechanisms underlying the toxic consequences relating this environmental toxicant and corneal injury. The protein targets identified using systems toxicology approach involving protein carbonylation needs further investigation and could potentially serve as biomarkers of CP-induced corneal toxicity.

Funding Information

This work was supported by a grant from the Associate Dean of Research Seed Grant Program, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver and (in part) by the Countermeasures Against Chemical Threats (CounterACT) Program, Office of the Director National Institutes of Health (OD) and the National Eye Institute (NEI), [Grant Number U01EY023143].

Abbreviations:

COX-2

cyclooxygenase-2

CP

chloropicrin

DMSO

dimethylsulfoxide

ER

endoplasmic reticulum

HCE

human corneal epithelial

HLE

human lung epithelial

HRPE

human retinal pigment epithelial

HO-1

heme oxygenase-1

JNK

jun-N terminal Kinase

MAPKs

Mitogen-activated protein kinases

MTT

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

NAC

N-Acetyl-L-Cysteine

4-HNE

4-Hydroxy-2-nonenal

PARP

poly (ADP-ribose) polymerase

PPP

pentose phosphate pathway

ROS

reactive oxygen species

Footnotes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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