Carbonyl chloride (phosgene) is a toxic industrial compound widely used in industry for the production of synthetic products, such as polyfoam rubber, plastics, and dyes. Exposure to phosgene results in a latent (1−24 h), potentially life-threatening pulmonary edema and irreversible acute lung injury. A genomic approach was utilized to investigate the molecular mechanism of phosgene-induced lung injury. CD-1 male mice were exposed whole body to either air or a concentration × time amount of 32 mg/m3 (8 ppm) phosgene for 20 min (640 mg × min/m3). Lung tissue was collected from air- or phosgene-exposed mice at 0.5, 1, 4, 8, 12, 24, 48, and 72 h postexposure. RNA was extracted from the lung and used as starting material for the probing of oligonucleotide microarrays to determine changes in gene expression following phosgene exposure. The data were analyzed using principal component analysis to determine the greatest sources of data variability. A three-way analysis of variance based on exposure, time, and sample was performed to identify the genes most significantly changed as a result of phosgene exposure. These genes were rank ordered by p values and categorized based on molecular function and biological process. Some of the most significant changes in gene expression reflect changes in glutathione synthesis and redox regulation of the cell, including upregulation of glutathione S-transferase α-2, glutathione peroxidase 2, and glutamate-cysteine ligase, catalytic subunit (also known as γ-glutamyl cysteine synthetase). This is in agreement with previous observations describing changes in redox enzyme activity after phosgene exposure. We are also investigating other pathways that are responsive to phosgene exposure to identify mechanisms of toxicity and potential therapeutic targets.

Background & Aims MicroRNA (miRNA) is highly stable in biospecimens and provides tissue-specific profiles, making it a useful biomarker of carcinogenesis. We aimed to discover a set of miRNAs that could accurately discriminate Barrett’s esophagus (BE) from normal esophageal tissue and to test its diagnostic accuracy when applied to samples collected by a non-invasive esophageal cell sampling device. Methods We analyzed miRNA expression profiles of 2 independent sets of esophageal biopsy tissues collected during endoscopy from 38 patients with BE tissues and 90 patients with non-BE esophagus (controls) using Agilent microarray and Nanostring counter assays. Consistently upregulated miRNAs were quantified by real-time PCR in esophageal tissues collected by Cytosponge from patients with BE or without BE. miRNAs from plasmids and anti-sense oligonucleotides were expressed in NES normal esophageal squamous cells and effects on proliferation and gene expression patterns were analyzed. Results We identified 15 miRNAs that were significantly upregulated in BE vs control tissues. Of these, 11 (MIR215, MIR194, MIR 192, MIR196a, MIR199b, MIR10a, MIR145, MIR181a, MIR30a, MIR7, MIR199a) were validated in Cytosponge samples. The miRNAs with the greatest increases in BE tissues (7.9-fold increase inexpression or more, P<0.0001: MIR196a, MIR192, MIR194, and MIR215) each identified BE vs control tissues with area under the curve (AUC) values of 0.82 or more. We developed an optimized multivariable logistic regression model based on expression levels of 6 miRNAs (MIR7, MIR30a, MIR181a, MIR192, MIR196a, and MIR199a) that identified patients with BE with an AUC value of 0.89, 86.2% sensitivity, and 91.6% specificity. Expression level of MIR192, MIR196a, MIR199a, combined with Trefoil Factor 3 (TFF3), identified patients with BE with an AUC of 0.93, 93.1% sensitivity, and 93.7% specificity. Hypo-methylation was observed in the promoter region of the highly upregulated cluster MIR192-194. Overexpression of these miRNAs in NES cells increased their proliferation, via GRHL3 and PTEN signaling. Conclusions In analyses of miRNA expression patterns of BE vs non-BE tissues, we identified a profile that can identify Cytosponge samples from patients with BE with an AUC of 0.93. Expression of MIR194 is increased in BE samples via epigenetic mechanisms that might be involved in BE pathogenesis.

Background The tissue-specificity and robustness of miRNAs may aid risk prediction in individuals diagnosed with Barrett’s esophagus. As a initial step, we assessed whether miRNAs can positively distinguish esophageal adenocarcinoma (EA) from the precursor metaplasia Barrett’s esophagus (BE). Methods In a case-control study of 150 EAs frequency-matched to 148 BE cases, we quantitated expression of 800 human miRNAs in FFPE tissue RNA using NanoString miRNAv2. We tested differences in detection by case group using the chi-square test and differences in expression using the Wilcoxon rank-sum test. Bonferroni-corrected statistical significance threshold was set at P<6.25E–05. Sensitivity and specificity were assessed for the most significant miRNAs using five-fold cross-validation. Results We observed 46 distinct miRNAs significantly increased in EA compared with BE; 35 of which remained when restricted to T1b and T2 malignancies. Three miRNAs (miR-663b, miR-421, miR-502-5p) were detected in >80% EA, but <20% of BE. Seven miRNAs (miR-4286, miR-630, miR-575, miR-494, miR-320e, miR-4488, miR-4508) exhibited the most extreme differences in expression with >5 fold-increases. Using five-fold cross validation, we repeated feature (miR) selection and case-control prediction and computed performance criteria. Each of the five folds selected the same top ten miRs which, together, provided 98% sensitivity and 95% specificity. Conclusion This study provides evidence that tissue miRNA profiles can discriminate EA from BE. This large analysis has identified miRNAs that merit further investigation in relation to pathogenesis and diagnosis of EA. Impact These candidate miRNAs may provide a means for improved risk stratification and more cost-effective surveillance.

Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent alkylating agent. Three treatment compounds have been shown to limit SM damage in the mouse ear vesicant model: dimercaprol, octyl homovanillamide, and indomethacin. Microarrays were used to determine gene expression profiles of biopsies taken from mouse ears after exposure to SM in the presence or absence of treatment compounds. Mouse ears were topically exposed to SM alone or were pretreated for 15 min with a treatment compound and then exposed to SM. Ear tissue was harvested 24 h after exposure for ear weight determination, the endpoint used to evaluate treatment compound efficacy. RNA extracted from the tissues was used to generate microarray probes for gene expression profiling of therapeutic responses. Principal component analysis of the gene expression data revealed partitioning of the samples based on treatment compound and SM exposure. Patterns of gene responses to the treatment compounds were indicative of exposure condition and were phenotypically anchored to ear weight. Pretreatment with indomethacin, the least effective treatment compound, produced ear weights close to those treated with SM alone. Ear weights from animals pretreated with dimercaprol or octyl homovanillamide were more closely associated with exposure to vehicle alone. Correlation coefficients between gene expression level and ear weight revealed genes involved in mediating responses to both SM exposure and treatment compounds. These data provide a basis for elucidating the mechanisms of response to SM and drug treatment and also provide a basis for developing strategies to accelerate development of effective SM medical countermeasures.Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent bifunctional alkylating agent capable of modifying and crosslinking cellular macromolecules such as DNA and protein by nucleophilic attack (Papirmeister et al., 1991). SM exposure can produce debilitating pulmonary, ocular, and cutaneous injuries. After cutaneous exposure to SM, there is a dosedependent latent phase of 8 to 24 h that precedes clinical expression of tissue damage. Erythema occurs initially and is followed by vesication because of separation at the epidermal-dermal junction. This results in large fluid-filled lesions that are long-lasting and slow to heal (Papirmeister et al., 1991;Petrali and Oglesby-Megee, 1997). The formation of blisters is accompanied by a potent inflammatory response, observed as increased production of inflammatory mediators and infiltration of the exposure area by activated immune cells (Rikimaru et al

No abstract