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

doi:10.1016/j.toxlet.2019.12.023

Toxicology Letters

Volume 322, 1 April 2020, Pages 1-11

https://doi.org/10.1016/j.toxlet.2019.12.023 Get rights and content

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.

Graphical abstract

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.

Section snippets

Materials and methods

Chemicals and Reagents.CP, N-Acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), anti-beta-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 Jun-amino-terminal kinase (JNK1/2; Thr183/ Tyr185), JNK1/2, phosphorylated p38 (Thr180/Tyr182), p38,

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

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

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].

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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¹: Current Address: Department of Molecular and Translational Medicine, Texas Tech University Health Sciences Center, El Paso, 79905, Texas, USA.

²: Current Address: Lions Eye Institute for Transplant & Research, Tampa, Florida, 33605, USA.

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

Abstract

Graphical abstract

Introduction

Section snippets

Materials and methods

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

Discussion

Funding Information

Declaration of Competing Interest

Mol. Cell

Clin. Chim. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Kidney Res. Clin. Pract.

Free Radic. Biol. Med.

Toxicol. Lett.

Toxicol. Lett.

Toxicology in vitro: an international journal published in association with BIBRA

Free Radic. Biol. Med.

J. Biol. Chem.

Acute exposure guideline levels

Chloropicrin.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Cancer Res.

Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells

Cancer Res.

Gene Ontology: tool for the unification of biology

Nat. Genet.

Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal

Oxid. Med. Cell. Longev.

Oxidative stress and lipid peroxidation products in Cancer progression and therapy

ISRN Oncol.

Community exposure following a drip-application of chloropicrin

J. Agromedicine

Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer

Pharmacol. Rev.

Biodehalogenation. The metabolism of chloropicrin by Pseudomonas sp

J. Agric. Food Chem.

Comparative proteomic analysis of carbonylated proteins from the striatum and cortex of pesticide-treated mice

Parkinsons Dis.

DNA Repair and Mutagenesis

Chlorine: state of the art

Lung

Heme Oxygenase-1, a key enzyme for the cytoprotective actions of halophenols by upregulating Nrf2 expression via activating Erk1/2 and PI3K/Akt in EA.hy926 cells

Oxid. Med. Cell. Longev.

4-Hydroxynonenal inhibits SIRT3 via thiol-specific modification

Chem. Res. Toxicol.