Considerations for the optimization of in vitro models of chloropicrin toxicity.

Chloropicrin (CP) is a common agricultural fumigant historically used as a chemical warfare agent and is a concern for potential use in warfare and terrorist applications. Our inability to effectively treat CP-induced injuries makes it essential to better understand CP toxicity. We set out to elucidate variables that must be understood to achieve optimal exposure conditions for in vitro investigations given that such models are important for the study of CP injury and potential therapeutics. To this end, we evaluated the effects of volatility, cell seeding density, and serum concentration of cell culture medium on CP toxicity in an immortalized human corneal epithelial cell line. We found that even with very dilute solutions, CP remained highly volatile, so much so that a 0.0019% CP solution resulted in 90% cell death at time 0, but was nearly nontoxic 45 min later. Not surprisingly, the CP-induced IL-8 response was shown to vary with cell viability in this experiment. After exposure with 0.00115% CP, cells that were 12% confluent experienced over 40% more cell death than cells exposed at 87% confluency. Exposure with the same CP dose in medium containing concentrations of fetal bovine serum (FBS) ranging from 0.1% to 15% exhibited a 17% difference in cell viability. Given that chemical toxicity can be significantly influenced by volatility, cell density, and serum content of cell culture medium, these phenomena should be explored during the development and optimization of toxicant exposure models.

siRNA high throughput screening identifies regulators of chloropicrin and hydrogen fluoride injury in human corneal epithelial cell models.

Corneal injuries induced by various toxicants result in similar clinical presentations such as corneal opacity and neovascularization. Many studies suggest that several weeks post-exposure a convergence of the molecular mechanisms drives these progressive pathologies. However, chemical agents vary in toxicological properties, and early molecular responses are anticipated to be somewhat dissimilar for different toxicants. We chose 3120 targets from the Dharmacon Human Druggable genome to screen for chloropicrin (CP) and hydrogen fluoride (HF) corneal injury as we hypothesized that targets identified in vitro may be effective as therapeutic targets in future studies. Human immortalized corneal epithelial cells (SV40-HCEC) were used for screening. Cell viability and IL-8 were analyzed to down-select hits into validation studies, where multiplex cytokine analysis and high content analysis were performed to understand toxicant effect and target function. Some endpoints were also evaluated in a second human immortalized corneal epithelial cell line, TCEpi. Over 20 targets entered validation studies for CP and HF; of these, only three targets were shared: NR3C1, RELA, and KMT5A. These findings suggest that early molecular responses to different toxicants may be somewhat distinctive and present dissimilar targets for possible early intervention.

Development of mouse models for the study of chloropicrin and hydrogen fluoride ocular injury.

The possibility of chemical terrorism within the United States is a rising concern, with the eye being one of the most sensitive tissues to toxicant exposure. We sought to develop mouse models of toxicant-induced ocular injury for the purpose of evaluating potential therapeutics. Chloropicrin (CP) and hydrogen fluoride (HF) were selected for the study owing to their reportedly high potential to induce ocular injury. Eyes of female BALB/c mice were exposed to CP or HF vapor in order to produce a moderate injury, as defined by acute corneal epithelial loss followed by progressive corneal pathology with the absence of injury to deeper eye structures. Clinical injury progression was evaluated up to 12 weeks postexposure, where a significant dose-dependent induction of corneal neovascularization was measured. Histopathology noted epithelial necrosis and stromal edema as early as 24 h after exposure but was resolved by 12 weeks. A significant increase in inflammatory cytokine concentrations was measured in the cornea 24 h after exposure and returned to baseline by day 14. The ocular injury models we developed here for CP and HF exposure should serve as a valuable tool for the future evaluation of novel therapeutics and the molecular mechanisms of injury.

Evaluation of a Synthetic Bedding Substrate for Mice (Mus musculus).

This study compared a synthetic bedding substrate (SBS), which has the potential to be a particulate-free animal bedding system, with the standard woodchip bedding. The objective was to demonstrate that the SBS is habitable for mice and reduces particulates to levels that would not contaminate the eye or potentially induce ocular (corneal) injury. Newly weaned mice were placed in either standard woodchip bedding or SBS. All mice were monitored regarding overall health (appearance, food and water intake, natural behavior, clinical signs, and provoked behavior) to verify their ability to adjust to the bedding. At 8 to 10 wk of age, the mice underwent slit-lamp evaluation for ocular (corneal) abnormalities. Results showed significant differences in body weight and overall health between bedding groups. The incidence of ocular abnormalities did not differ significantly between groups. We conclude that, without modifications and more testing, SBS is not a favorable bedding for mice, and results were inconclusive regarding its use as a bedding to preclude ocular contamination.

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

Identification and validation of vesicant therapeutic targets using a high-throughput siRNA screening approach.

Sulfur mustard [SM, bis-(2-chloroethyl) sulfide] is a highly reactive bifunctional alkylating agent that has been used as a vesicating agent in warfare scenarios to induce severe lung, skin, and eye injury. SM cutaneous lesions are characterized by both vesication and severe inflammation, but the molecular mechanisms that lead to these signs and symptoms are not well understood. There is a pressing need for effective therapeutics to treat this injury. An understanding of the molecular mechanisms of injury and identification of potential therapeutic targets is necessary for rational therapeutic development. We have applied a high-throughput small interfering RNA (siRNA) screening approach to the problem of SM cutaneous injury in an effort to meet these needs. Our siRNA screening efforts have initially focused on SM-induced inflammation in cutaneous injury since chronic inflammation after exposure appears to play a role in progressive clinical pathology, and intervention may improve clinical outcome. Also, targets that mitigate cellular injury should reduce the inflammatory response. Historical microarray data on this injury were mined for targets and pathways implicated in inflammation, and a siRNA library of 2,017 targets was assembled for screening. Primary screening and library deconvolution were performed using human HaCaT keratinocytes and focused on cell death and inflammatory markers as end points. Using this in vitro approach, we have identified and validated novel targets for the potential treatment of SM-induced cutaneous injury.

Association of the α(2)δ(1) subunit with Ca(v)3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes.

Voltage-sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α(1) pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs and a relatively small amount of L-type α(1) subunits. Auxiliary VSCC subunits have several functions, including modulating gating kinetics, trafficking of the channel, and phosphorylation events. The influence of the α(2)δ auxiliary subunit on T-type VSCCs and the physiologic consequences of that association are incompletely understood and have yet to be investigated in bone. In this study we postulated that the auxiliary α(2) δ subunit of the VSCC complex modulates mechanically regulated ATP release in osteocytes via its association with the T-type Ca(v) 3.2 (α(1H) ) subunit. We demonstrated by reverse-transcriptase polymerase chain reaction, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type Ca(v)3.2(α(1H)) subunit more abundantly than the L-type Ca(v)1.2 (α(1C)) subunit. We also demonstrated that the α(2) δ(1) subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Ca(v)3.2 (α(1H)) subunit in MLO-Y4 cells. Interestingly, siRNA-mediated knockdown of α(2) δ(1) completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α(2)δ(1) subunit resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α(2)δ(1) knockdown showed decreased membrane localization of Ca(v) 3.2 (α(1H)) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Ca(v) 3.2 (α(1H)) at the cell membrane.

Sulfur mustard induced cytokine production and cell death: investigating the potential roles of the p38, p53, and NF-kappaB signaling pathways with RNA interference.

Cutaneous and ocular injuries caused by sulfur mustard (SM; bis-(2-chloroethyl) sulfide) are characterized by severe inflammation and death of exposed cells. Given the known roles of p38MAPK and NF-kappaB in inflammatory cytokine production, and the known roles of NF-kappaB and p53 in cell fate, these pathways are of particular interest in the study of SM injury. In this study, we utilized inhibitory RNA (RNAi) targeted against p38 alpha, the p50 subunit of NF-kappaB, or p53 to characterize their role in SM-induced inflammation and cell death in normal human epidermal keratinocytes (NHEK). Analysis of culture supernatant from 200 microM SM-exposed cells showed that inflammatory cytokine production was inhibited by p38 alpha RNAi but not by NF-kappaB p50 RNAi. These findings further support a critical role for p38 in SM-induced inflammatory cytokine production in NHEK and suggest that NF-kappaB may not play a role in the SM-induced inflammatory response of this cell type. Inhibition of NF-kappaB by p50 RNAi did, however, partially inhibit SM-induced cell death, suggesting a role for NF-kappaB in SM-induced apoptosis or necrosis. Interestingly, inhibition of p53 by RNAi potentiated SM-induced cell death, suggesting that the role of p53 in SM injury, may be complex and not simply prodeath.

Signaling molecules in sulfur mustard-induced cutaneous injury.

OBJECTIVE: Sulfur mustard (SM) is a potent alkylating agent that can induce severe cutaneous injury. Though much is known regarding the gross pathology of SM injury, the molecular and cellular basis for this pathology is not well understood. General cellular processes such as inflammation, DNA damage response, and apoptosis have been hypothesized to be involved in SM injury. However, the specific molecules, signaling pathways, and gene products involved in the pathogenesis of SM injury have not been elucidated. This review discusses the molecular mechanisms observed in in vivo and in vitro models of cutaneous SM injury. METHODS: The historical literature on the clinical pathology of SM-induced cutaneous injury is summarized, and recent work elucidating molecular signaling pathways involved in SM toxicity is extensively reviewed. In addition, this review focuses the discussion of SM-induced molecular mechanisms on those that have been experimentally validated in models of SM injury. RESULTS: Recent work has uncovered potential roles for a number of signaling molecules. In particular, molecules in inflammatory signaling, DNA damage response, apoptosis signaling, and calcium signaling have been implicated in SM injury. These include signaling molecules involved in inflammation (e.g. p38 MAP kinase), apoptosis (e.g. p53, NF-kappa B, caspases, Fas), and cell stress responses (e.g. calcium, calmodulin). CONCLUSIONS: Many of the molecules and mechanisms implicated in SM injury are now being experimentally validated. Critical questions are proposed that remain to be answered to increase our understanding of SM toxicity and accelerate the development of vesicant therapeutics.