Countermeasures against Pulmonary Threat Agents.

Inhaled toxicants are used for diverse purposes, ranging from industrial applications such as agriculture, sanitation, and fumigation to crowd control and chemical warfare, and acute exposure can induce lasting respiratory complications. The intentional release of chemical warfare agents (CWAs) during World War I caused life-long damage for survivors, and CWA use is outlawed by international treaties. However, in the past two decades, chemical warfare use has surged in the Middle East and Eastern Europe, with a shift toward lung toxicants. The potential use of industrial and agricultural chemicals in rogue activities is a major concern as they are often stored and transported near populated areas, where intentional or accidental release can cause severe injuries and fatalities. Despite laws and regulatory agencies that regulate use, storage, transport, emissions, and disposal, inhalational exposures continue to cause lasting lung injury. Industrial irritants (e.g., ammonia) aggravate the upper respiratory tract, causing pneumonitis, bronchoconstriction, and dyspnea. Irritant gases (e.g., acrolein, chloropicrin) affect epithelial barrier integrity and cause tissue damage through reactive intermediates or by direct adduction of cysteine-rich proteins. Symptoms of CWAs (e.g., chlorine gas, phosgene, sulfur mustard) progress from airway obstruction and pulmonary edema to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which results in respiratory depression days later. Emergency treatment is limited to supportive care using bronchodilators to control airway constriction and rescue with mechanical ventilation to improve gas exchange. Complications from acute exposure can promote obstructive lung disease and/or pulmonary fibrosis, which require long-term clinical care. SIGNIFICANCE STATEMENT: Inhaled chemical threats are of growing concern in both civilian and military settings, and there is an increased need to reduce acute lung injury and delayed clinical complications from exposures. This minireview highlights our current understanding of acute toxicity and pathophysiology of a select number of chemicals of concern. It discusses potential early-stage therapeutic development as well as challenges in developing countermeasures applicable for administration in mass casualty situations.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

BACKGROUND: Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. RESULTS: We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 µM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. CONCLUSION: Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Pathogenesis of phosgene poisoning.

Phosgene inhalation in concentrations greater than 1 ppm may produce a transient bioprotective vagus reflex with rapid shallow breathing in some individuals. Phosgene concentrations greater than 3 ppm are moderately irritating to eyes and upper airways. Toxic phosgene doses (greater than or equal to 30 ppm X min) inhaled into the terminal respiratory passages render the blood-air-barrier more permeable to blood plasma, which gradually collects in the lung. Some time passes, however, until the collection of fluid provokes signs and symptoms. This period in which the patient experiences relative well-being is known as the clinical latent phase. The clinical symptoms which follow and the pathological changes underlying them are discussed in detail; dose-effect relationships are demonstrated. The regression phase after poisoning has been overcome is briefly sketched.

Inactivation of cathepsin D by dithiophosgene and by 2,2-dichloro-1,3-dithiacyclobutanone.

Cathepsin D, purified from bovine spleen, is inactivated by the unstable inhibitors dithiophosgene and 2,2-dichloro-1,3-dithiacyclobutanone. Inhibition constants are identical for both of the compounds tested: Ki 96.1 muM;k/c0.406. It appears that the active species is 2,2-dichloro-1,3-dithiacyclobutanone, to which dithiophosgene is hydrolysed before cathepsin D inactivation.

Response of the pulmonary surfactant system to phosgene.

Rats were exposed to 240 ppm X min phosgene (1.0 ppm for 4 hrs) in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were removed for determination of wet weight; total, microsomal and surfactant protein concentrations; surfactant phospholipid concentrations; and 1-acyl-2-lyso-phosphatidylcholine: palmitoyl-CoA acyl transferase activity. Immediately upon termination of the phosgene exposure, microsomal protein and acyl transferase activity were reduced below, and lung wet weight was elevated above, control levels. From Day 1 through Day 3 after the exposure, all measured parameters, except for the phosphatidylinositol constituent of the surfactant fraction, were increased above the control values. In general, maximum levels were observed on Day 2; however, the acyl transferase activity and surfactant concentration continued to increase on Day 3. The results suggest components of the pulmonary surfactant system may be involved in maintenance of pulmonary fluid balance and the presence of excess water in the lungs as a result of phosgene exposure may represent a signal for increased synthesis of anti-edematogenic materials in order to promote removal of the inappropriate fluid.

In vitro adduct formation of phosgene with albumin and hemoglobin in human blood.

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [(14)C]phosgene (0-750 microM), a significant part of radioactivity (0-13%) became associated with globin and albumin. Upon Pronase digestion of globin, one of the adducts was identified as the pentapeptide O=C-(V-L)-S-P-A, representing amino acid residues 1-5 of alpha-globin, with a hydantoin function between N-terminal valine and leucine. Micro-LC/tandem MS analyses of tryptic as well as V8 protease digests identified one of the adducts to albumin as a urea resulting from intramolecular bridging of lysine residues 195 and 199. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with multiple-reaction monitoring (MRM), enabling the detection in human blood of an in vitro exposure level of >/=1 microM phosgene.

Response of pulmonary energy metabolism to phosgene.

Rats were exposed to phosgene at a concentration of 1.0 ppm for 4 hours in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were obtained for histological and biochemical assessments. Edema was estimated by histological examination and by measurement of lung wet and dry weights. In parallel studies, pulmonary mitochondrial respiratory activity was measured using Clark oxygen electrodes. The significant reduction in respiratory control index (State 3 respiration/State 4 respiration) found immediately following phosgene exposure coincided with the highest level of % lung water. There was a concomitant decrease of ATP concentration that persisted on the third day after exposure. Na-K-ATPase activity was reduced 1 day after exposure, thus a lowered ATP level preceded a reduction in Na-K-ATPase or sodium pump activity. The reduction in ATP level and Na-K-ATPase activity may play a major role in damage to lung tissue following exposure to phosgene.