Medical Aspects of Chemical Warfare

The first-aid treatment of mass casualties from nerve gas relies mainly upon the use of drugs, and provision for their self-injection is recommended. Means for giving artificial respiration must also be provided, even though its large-scale use is regarded as impracticable. Prophylactic oxime (2 g. PAM chloride orally) is recommended if the situation permits. Some nerve gases are extremely rapid in action, and following exposure (or suspicion of exposure) 4 mg. of atropine and 2 g. of PAM chloride should be injected intramuscularly without delay. Preferably, atropine should be given intravenously. At the same time any clothing contaminated with liquid nerve gas should be removed and the skin cleansed thoroughly with a suitable fluid. Following this, the casualty should be watched closely for one hour. If poisoning develops despite these measures, or is already established, injection of atropine should be continued at short intervals until improvement occurs.     

Predator-Prey Chemical Warfare Determines the Expression of Biocontrol Genes by Rhizosphere-Associated Pseudomonas fluorescens

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.Bacterial communities are heavily consumed by protozoan predators (30), and predation is a major force shaping the structure of microbial communities in both aquatic and terrestrial ecosystems (34, 35). The competitiveness of bacteria strongly depends on their ability to avoid predation (9, 22), and many species have developed defense mechanisms such as the production of toxins, which reduces mortality by repelling or killing predators (21, 24). Toxin production, however, is energetically costly, and defense theory predicts that prey species should optimize the investment in defense according to the resources available and the predation risk (40), for example, in response to predator-associated chemical cues (4, 15). In bacteria the production of defense traits is tightly regulated by various sensor cascades (11), and defense mechanisms, such as the formation of inedible morphotypes or microcolonies, can be elicited in the presence of predators (45).Toxin production is one of the most powerful defense strategies, and in the present study we tested whether bacteria can also modulate the production of toxic secondary metabolites in response to protozoan predators. We investigated the chemical communication between the soil bacterium Pseudomonas fluorescens CHA0 and the bacterivorous amoeba Acanthamoeba castellanii, a ubiquitous soil protist feeding on a wide range of bacterial species (33). P. fluorescens CHA0 effectively colonizes the rhizosphere of plants and produces various extracellular toxins including pyrrolnitrin (PRN), 2,4-diacetylphloroglucinol (DAPG), hydrogen cyanide (HCN), and pyoluteorin (PLT) (18). These toxins reduce predation pressure and enhance the competitiveness of the bacteria in the rhizosphere (22) but also act antagonistically against plant pathogens, thereby promoting plant growth (11).The production of secondary metabolites by P. fluorescens is a dynamic process that depends on environmental factors, such as nutrient availability (12), cell density (18), or the presence of phytopathogens (27). We hypothesized that P. fluorescens is also able to sense predators and responds by increasing the expression of toxin genes.Predators or competitors susceptible to toxins can adopt counterstrategies to repress their production. For example, the fungal pathogen Fusarium can inhibit the production of DAPG by pseudomonads (26), and we hypothesized that A. castellanii can counteract prey defense by inhibiting bacterial toxin production.We investigated the effects of predator-prey interactions on the regulation of the production of the extracellular toxins DAPG, PLT, PRN, and HCN using a set of autofluorescent green fluorescent protein (GFP)- and mCherry-based reporter fusions (2, 32). Autofluorescent reporter fusions allow in situ measurement of gene expression patterns and have been applied to monitor the expression of antifungal genes in the rhizosphere (10, 32). The response of the bacteria to predators or associated signal molecules was investigated in batch experiments and on barley roots.     

Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O₂ chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (f_V) maximally at 100 μM (EC₅₀ = 24.5 μM). Hypoxia elevated f_V from 93.8 to 145.3 breaths min^-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced f_V at 50 μM (EC₅₀ = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O₂-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O₂ sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.     

Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX

Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.     

Antiprion Drugs as Chemical Tools to Uncover Mechanisms of Prion Propagation

A number of drugs active against prions either in vitro, in cellular systems or in vivo in animal models have been isolated in various screening assays. In this minireview, we would like to suggest, that in addition to their direct interest as potential therapeutic agents, these molecules could be used as original research tools to understand prion propagation. The use of antiprion compounds as tool to understand fundamentals of prion propagation relies on reverse screening approaches. These global genetic and/or biochemical approaches aim to identify the intracellular target(s) and mechanism of action of the drugs. Once those are known, the biological activity of the compounds can be optimized on a rational basis, their potential side effects understood and minimized. In vitro enzyme-based screening assays can then be designed to allow discovery of new, more potent and selective molecules. Here we describe the main comprehensive biochemical and genetical approaches to realize reverse screening approaches based on antiprion drugs. We will finish by discussing the interest of using drug inactivation of specific targets as a substitute to genetic inactivation.Key Words: prion, amyloid fibers, protein folding, protein chaperone, antiprion drugs, reverse screeningA number of drugs have been isolated as active against mammalian prion (reviewed in ref. ¹). For most of these molecules, the mode of action and targets remain largely unknown. In principle, two main modes of action for antiprion drugs can be envisioned: either in cis directly on PrP^C/PrP^Sc, or in trans by interfering with the activity of cellular factors required for prion propagation. Some compounds are thought to bind directly to PrP^C or PrP^Sc (action in cis). Among these compounds are Congo Red (CR), Pentosan Polysulfate (PPS) or Glycosaminoglycans (GAGs). Other compounds are thought to act in trans by affecting PrP^C or PrP^Sc indirectly. Among these molecules are various lysosomotropic factors including the antimalarial drugs Quinacrine (QC) and Chloroquine. Indeed, the lysosome is a potential site of conversion of PrP^C to PrP^Sc.² In addition, a recent report,³ proposes that QC''s antiprion activity is related to its ability to redistribute cholesterol from the plasma membrane to intracellular compartments, thereby destabilizing membrane domains. This conclusion was drawn from correlation experiments indicating that drugs known to display cholesterol-redistributing activity (but structurally unrelated to QC) also have antiprion potency. Finally, QC was also shown to interact directly with PrP.^4,5 The example of QC, with these conflicting results, thus illustrates the need for unbiaised and global approaches not driven by a preconcieved hypothesis on the drug mode of action. To our knowledge, no such approaches were applied for any of the known antiprion drugs, with the noticeable exception of chlorpromazine for which a haploinsufficiency profiling screen (HIP, see in the later section) has been published.⁶ In this minireview, we discuss the various advantages of defining extensively intracellular targets of antiprion drugs. We then would like to present some global approaches that can be applied to uncover, in an unbiaised manner, cellular mechanism(s) of action of compounds active against prions. To finish we propose that, once intracellular targets have been clearly identified, the drugs can be used to do “chemical genetics” to inactivate cellular target(s) which could be especially useful in situations where classical inactivation by mutagenesis is unpractical (for instance if redundant multigenic families are involved).