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.

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.