The prediction of hydrolysis and biodegradation of organophosphorus-based chemical warfare agents (G-series and V-series) using toxicology in silico methods.

Nerve agents (G- and V-series) are a group of extremely toxic organophosphorus chemical warfare agents that we have had the opportunity to encounter many times on a massive scale (Matsumoto City, Tokyo subway and Gulf War). The threat of using nerve agents in terrorist attacks or military operations is still present, even with establishing the Chemical Weapons Convention as the legal framework. Understanding their environmental sustainability and health risks is critical to social security. Due to the risk of contact with dangerous nerve agents and animal welfare considerations, in silico methods were used to assess hydrolysis and biodegradation safely. The environmental fate of the examined nerve agents was elucidated using QSAR models. The results indicate that the investigated compounds released into the environment hydrolyse at a different rate, from extremely fast (<1 day) to very slow (over a year); V-agents undergo slower hydrolysis compared to G-agents. V-agents turned out to be relatively challenging to biodegrade, the ultimate biodegradation time frame of which was predicted as weeks to months, while for G-agents, the overwhelming majority was classified as weeks. In silico methods for predicting various parameters are critical to preparing for the forthcoming application of nerve agents.

No-observed-adverse-effect-level (NOAEL) assessment as an optimized dose of cholinesterase reactivators for the treatment of exposure to warfare nerve agents in mice.

Despite the international convention on the prohibition of chemical weapons ratified in 1997, the threat of conflicts and terrorist attacks involving such weapons still exists. Among these, organophosphorus-nerve agents (OPs) inhibit cholinesterases (ChE) causing cholinergic syndrome. The reactivation of these enzymes is therefore essential to protect the poisoned people. However, these reactivating molecules, mainly named oximes, have major drawbacks with limited efficacy against some OPs and a non-negligible ChE inhibitor potential if administered at an inadequate dose, an effect that they are precisely supposed to mitigate. As a result, this project focused on assessing therapeutic efficacy, in mice, up to the NOAEL dose, the maximum dose of oxime that does not induce any observable toxic effect. NOAEL doses of HI-6 DMS, a reference oxime, and JDS364. HCl, a candidate reactivator, were assessed using dual-chamber plethysmography, with respiratory ventilation impairment as a toxicity criterion. Time-course modeling parameters and pharmacodynamic profiles, reflecting the interaction between the oxime and circulating ChE, were evaluated for treatments at their NOAEL and higher doses. Finally, the therapeutic potential against OPs poisoning was determined through the assessment of protective indices. For JDS364. HCl, the NOAEL dose corresponds to the smallest dose inducing the most significant therapeutic effect without causing any abnormality in ChE activity. In contrast, for HI-6 DMS, its therapeutic benefit was observed at doses higher than its NOAEL, leading to alterations in respiratory function. These alterations could not be directly correlated with ChE inhibition and had no adverse effects on survival. They are potentially attributed to the stimulation of non-enzymatic cholinergic targets by HI-6 DMS. Thus, the NOAEL appears to be an optimal dose for evaluating the efficacy of oximes, particularly when it can be linked to respiratory alterations effectively resulting from ChE inhibition.

The prediction of hydrolysis and biodegradation of Novichoks using in silico toxicology methods.

Novichoks constitute a relatively new class of nerve agents of extreme toxicity that we have had the opportunity to experience three times already. After the first case (Salisbury, UK), a public debate about Novichoks began, which resulted in the realisation of the nature of these chemicals. From a social security point of view, examining their properties, especially toxicological and environmental aspects, are crucial. After the CWC (Chemical Warfare Agent) list update, the candidate structures for the Novichoks may be over 10,000 compounds. It would be extremely laborious to conduct experimental research for each. Understanding their environmental persistence and health hazards is an essential national issue. Moreover, due to the high risk posed by contact with hazardous Novichok substances, in silico research was applied to estimate hydrolysis and biodegradation safely. The present study elucidates, using QSAR models, the environmental fate of the Novichoks studied (n = 17). The results indicate that Novichoks released into the environment hydrolyse at various rates, from extremely fast (<1 day) to very slow (more than a year). Furthermore, ultimate biodegradation from weeks to months is expected for most compounds, which classifies them as relatively difficult biodegradable. Applying reliable in silico methods (QSAR Toolbox and EPI Suite) for predicting various parameters is crucial to prepare for the upcoming usage of Novichoks.

Establishment of a Novel Oral Murine Model of Ricin Intoxication and Efficacy Assessment of Ovine Ricin Antitoxins.

Ricin, produced from the castor beans of Ricinus communis, is a cytotoxin that exerts its action by inactivating ribosomes and causing cell death. Accidental (e.g., ingestion of castor beans) and/or intentional (e.g., suicide) exposure to ricin through the oral route is an area of concern from a public health perspective and no current licensed medical interventions exist to protect from the action of the toxin. Therefore, we examined the oral toxicity of ricin in Balb/C mice and developed a robust food deprivation model of ricin oral intoxication that has enabled the assessment of potential antitoxin treatments. A lethal oral dose was identified and mice were found to succumb to the toxin within 48 h of exposure. We then examined whether a despeciated ovine F(ab')2 antibody fragment, that had previously been demonstrated to protect mice from exposure to aerosolised ricin, could also protect against oral intoxication. Mice were challenged orally with an LD99 of ricin, and 89 and 44% of mice exposed to this otherwise lethal exposure survived after receiving either the parent anti-ricin IgG or F(ab')2, respectively. Combined with our previous work, these results further highlight the benefit of ovine-derived polyclonal antibody antitoxin in providing post-exposure protection against ricin intoxication.

Assessment of zebrafish embryo photomotor response sensitivity and phase-specific patterns following acute- and long-duration exposure to neurotoxic chemicals and chemical weapon precursors.

Zebrafish are an attractive model for chemical screening due to their adaptability to high-throughput platforms and ability to display complex phenotypes in response to chemical exposure. The photomotor response (PMR) is an established and reproducible phenotype of the zebrafish embryo, observed 24 h post-fertilization in response to a predefined sequence of light stimuli. In an effort to evaluate the sensitivity and effectiveness of the zebrafish embryo PMR assay for toxicity screening, we analyzed chemicals known to cause both neurological effects and developmental abnormalities, following both short (1 h) and long (16 h+) duration exposures. These include chemicals that inhibit aerobic respiration (eg, cyanide), acetyl cholinesterase inhibitors (organophosphates pesticides) and several chemical weapon precursor compounds with variable toxicity profiles and poorly understood mechanisms of toxicity. We observed notable concentration-responsive, phase-specific effects in the PMR after exposure to chemicals with a known mechanism of action. Chemicals with a more general toxicity profile (toxic chemical weapon precursors) appeared to reduce all phases of the PMR without a notable phase-specific effect. Overall, 10 of 20 chemicals evaluated elicited an effect on the PMR response and eight of those 10 chemicals were picked up in both the short- and long-duration assays. In addition, the patterns of response uniquely differentiated chemical weapon precursor effects from those elicited by inhibitors of aerobic respiration and organophosphates. By providing a rapid screening test for neurobehavioral effects, the zebrafish PMR test could help identify potential mechanisms of action and target compounds for more detailed follow-on toxicological evaluations. Approved for public release: distribution unlimited.

Assessment of exogenous melatonin action on mouse liver cells after exposure to soman.

Melatonin is a hormone with many different biological activities and therefore seems to be an important factor reducing the harmful effects caused by toxic organophosphorus compounds. In this study, we attempted to evaluate the protective effect of melatonin on liver cells of mice challenged with chemical warfare agent-soman. The study was conducted at the level of ultrastructural and biochemical changes (analysis of the activity of model lysosomal enzymes and assessment of the level of lipid peroxidation). Significant biochemical and ultrastructural changes were found in the studied mouse hepatocytes after administration of soman alone, and soman in combination with melatonin, and the scope of the disclosed changes was dependent on the time of action of the examined factors. Melatonin has shown protective action, shielding liver cells from toxic effects of soman, which may result from its antioxidant properties and stimulation of the lysosomal compartment, the system coordinating the isolation and removal of cell-threatening processes.

Assessment of mouse strain differences in baseline esterase activities and toxic response to sarin.

Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naïve mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure.

Dual-Use Nano-Neurotechnology: An assessment of the implications of trends in science and technology.

The chemical and biological nonproliferation regime stands at a watershed moment, when failure seems a real possibility. After the unsuccessful outcome of the 2016 Eighth Review Conference, the future of the Biological and Toxin Weapons Convention is uncertain. As the Chemical Weapons Convention (CWC) approaches its Fourth Review Conference in 2018, it has almost completed removing the huge stocks of chemical weapons, but it now faces the difficult organizational task of moving its focus to preventing the reemergence of chemical weapons at a time when the international security situation appears to be increasingly more difficult and dangerous. In this article, we assess the current and near-term state (5-10 years) and impact of three related areas of science and technology that could be of dual-use concern: targeted delivery of agents to the central nervous system (CNS), particularly by means of nanotechnology; direct impact of nanomaterials on synaptic functions in the CNS; and neuronal circuits in the brain that might be targeted by those with hostile intent. We attempt to assess the implications of our findings, particularly for the consideration of the problem of state-level interest in so-called nonlethal incapacitating chemical agents for law enforcement at the CWC Review Conference in 2018, but also more generally for the longer-term future of the chemical and biological nonproliferation regime.

Hydrogen sulphide and phosphine interactions with human skin in vitro.

Accidental or intentional releases of toxic gases can have significant public health consequences and emergency resource demands. Management of exposed individuals during hazardous material incidents should be risk and evidence based, but there are knowledge gaps in relation to dermal absorption of gases and management advice for potentially exposed individuals. Using a modified Organization for Economic Co-operation and Development (OECD) in vitro toxicology protocol with human donor skin, this article reports on two common and odorous chemicals, hydrogen sulphide and phosphine. Results show that undamaged human skin provides a good barrier to hydrogen sulphide (up to 800 ppm) and phosphine (up to 1000 ppm) penetration for up to 30 min exposures, with little variability in the presence of clothing or in elevated temperature and humidity conditions. A practical guideline template for skin decontamination has been developed, and implications of the research for first responders are outlined.

An easy method for the determination of active concentrations of cholinesterase reactivators in blood samples: Application to the efficacy assessment of non quaternary reactivators compared to HI-6 and pralidoxime in VX-poisoned mice.

Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 µmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice.

A dynamic approach for the impact of a toxic gas dispersion hazard considering human behaviour and dispersion modelling.

The release of toxic gases due to natural/industrial accidents or terrorist attacks in populated areas can have tragic consequences. To prevent and evaluate the effects of these disasters different approaches and modelling tools have been introduced in the literature. These instruments are valuable tools for risk managers doing risk assessment of threatened areas. Despite the significant improvements in hazard assessment in case of toxic gas dispersion, these analyses do not generally include the impact of human behaviour and people movement during emergencies. This work aims at providing an approach which considers both modelling of gas dispersion and evacuation movement in order to improve the accuracy of risk assessment for disasters involving toxic gases. The approach is applied to a hypothetical scenario including a ship releasing Nitrogen dioxide (NO2) on a crowd attending a music festival. The difference between the results obtained with existing static methods (people do not move) and a dynamic approach (people move away from the danger) which considers people movement with different degrees of sophistication (either a simple linear path or more complex behavioural modelling) is discussed.

Environmental genotoxicity assessment along the transport routes of chemical munitions leading to the dumping areas in the Baltic Sea.

The frequencies of micronuclei (MN), nuclear buds (NB) and nuclear buds on filament (NBf) were examined in 660 specimens of herring (Clupea harengus) collected in 2009-2014 at 65 study stations located mainly along the chemical munition transport routes in the Baltic Sea. The frequency of nuclear abnormalities was strongly increased in herring caught at four stations located close to chemical munition dumping sites, or CWAs - substances (chemical warfare agents) in sediments. Significant increase of MN, NB and NBf was observed in fish caught November 2010-2013 compared to 2009. The most significantly increased genotoxicity responses were recorded in fish caught at stations along CW (chemical weapons) transport routes, close to the Bornholm CW dumping area, in zones with CWAs in sediments and with oil-gas platforms.

Vapor inhalation exposure to soman in conscious untreated rats: preliminary assessment of neurotoxicity.

Neurological toxicity and brain injury following vapor inhalation exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in untreated non-anesthetized rats. In this study, male Sprague-Dawley rats (300-350 g) were exposed to 600 mg × min/m(3) of soman or vehicle in a customized head-out inhalation system for 7 min. Convulsant animals were observed for clinical signs and various regions of the brain (dorsolateral thalamus, basolateral amygdala, piriform cortex, and lateral cortex) were collected for pathological observations 24 h post-exposure. Signs of CWNA-induced cholinergic crises including salivation, lacrimation, increased urination and defecation, and tremors were observed in all soman-exposed animals. Soman-exposed animals at 24 h post-exposure lost 11% of their body weight in comparison to 2% in vehicle-exposed animals. Whole blood acetylcholinesterase (AChE) activity was significantly inhibited in all soman-exposed groups in comparison to controls. Brain injury was confirmed by the neurological assessment of hematoxylin-eosin (H&E) staining and microscopy in the piriform cortex, dorsolateral thalamus, basolateral amygdala, and lateral cortex. Severe damage including prominent lesions, edematous, congested, and/or hemorrhagic tissues was observed in the piriform cortex, dorsolateral thalamus, and lateral cortex in soman-exposed animals 24 h post-exposure, while only minimal damage was observed in the basolateral amygdala. These results indicate that inhalation exposure to soman vapor causes neurological toxicity and brain injury in untreated unanesthetized rats. This study demonstrates the ability of the described soman vapor inhalation exposure model to cause neurological damage 24 h post-exposure in rats.

Inhalation toxicity of soman vapor in non-anesthetized rats: a preliminary assessment of inhaled bronchodilator or steroid therapy.

Respiratory toxicity, injury and treatment following vapor inhalational exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in non-anesthetized rats. This study exposed male Sprague-Dawley rats (250-300g) to 520, 560, 600, 825 or 1410mg×min/m(3) of soman in a customized head-out inhalation system. Signs of CWNA-induced cholinergic crises were observed in all soman-exposed animals. The LCt50 of vaporized soman as determined by probit analysis was 593.1mg×min/m(3). All animals exposed to 825 and 1410mg×min/m(3) developed severe convulsions and died within 4-8min post-exposure. Edema measured by wet/dry weight ratio of the left lung lobe increased in a dose-dependent manner in all soman-exposed animals. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase (AChE) activities were inhibited dose-dependently in soman-exposed groups at 24h. A significant increase in total BAL protein was observed in soman-exposed animals at all doses. AChE activity was inhibited in lung and whole brain tissues in all soman-exposed animals. Histopathological analysis of the lungs of animals exposed to 600mg×min/m(3) of soman revealed prominent morphological changes including alveolar histiocytosis, hemorrhage and inflammation consisting of neutrophilic exudate. Exposure of animals to 600mg×min/m(3) of soman followed by treatment with two actuations for 10s of Combivent (21µg of ipratropium bromide and 120µg of albuterol sulfate) and Symbicort (80µg budesonide and 4.5µg formoterol) by inhalation into a modified metered dose inhaler (MDI) 10min post-exposure resulted in increased minute volume, but did not decrease mortality. These results indicate that inhalation exposure to soman vapor causes acute respiratory toxicity and injury in untreated, un-anesthetized rats and that inhalation treatment with Combivent or Symbicort did improve the respiratory outcomes, but did not influence lethality.

Functional genomic assessment of phosgene-induced acute lung injury in mice.

In this study, a genetically diverse panel of 43 mouse strains was exposed to phosgene and genome-wide association mapping performed using a high-density single nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was also used to improve the genetic resolution in the identification of genetic determinants of phosgene-induced acute lung injury (ALI). We prioritized the identified genes based on whether the encoded protein was previously associated with lung injury or contained a nonsynonymous SNP within a functional domain. Candidates were selected that contained a promoter SNP that could alter a putative transcription factor binding site and had variable expression by transcriptomic analyses. The latter two criteria also required that ≥10% of mice carried the minor allele and that this allele could account for ≥10% of the phenotypic difference noted between the strains at the phenotypic extremes. This integrative, functional approach revealed 14 candidate genes that included Atp1a1, Alox5, Galnt11, Hrh1, Mbd4, Phactr2, Plxnd1, Ptprt, Reln, and Zfand4, which had significant SNP associations, and Itga9, Man1a2, Mapk14, and Vwf, which had suggestive SNP associations. Of the genes with significant SNP associations, Atp1a1, Alox5, Plxnd1, Ptprt, and Zfand4 could be associated with ALI in several ways. Using a competitive electrophoretic mobility shift analysis, Atp1a1 promoter (rs215053185) oligonucleotide containing the minor G allele formed a major distinct faster-migrating complex. In addition, a gene with a suggestive SNP association, Itga9, is linked to transforming growth factor ß1 signaling, which previously has been associated with the susceptibility to ALI in mice.

Biosensor analysis of blood esterases for organophosphorus compounds exposure assessment: approaches to simultaneous determination of several esterases.

This paper reviews our previously published data and presents new results on biosensor assay of blood esterases. Tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. Experiments were performed on the quantitative determination of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), and neuropathy target esterase (NTE) in samples of whole blood of rats, mice, and humans. Good agreement was found between biosensor and spectrophotometric assays for AChE, BChE, and CaE. No direct comparison could be made for NTE because its activity cannot be measured spectrophotometrically in whole blood. A new method of simultaneous quantitative determination of AChE and BChE in test mixtures is also described. This method represents a bifunctional biosensor for the simultaneous analysis of choline and phenol based on integration of individual sensors. Algorithms for calculation of separate concentrations of AChE and BChE in the mixture were developed. The mean error of calculated component concentrations was approximately 6% for binary test mixtures. The present work provides a foundation for building multiplexed systems for the simultaneous determination of multiple esterases with applications to biomonitoring for exposures to organophosphorus compounds.

Consequence assessment of indoor dispersion of sarin--a hypothetical scenario.

The objective of this study is to provide a hypothetical scenario of indoor dispersion of the highly toxic nerve agent sarin in a large building, which can be used as a starting point for discussion, planning, training and exercises for emergency services and responsible authorities. The indoor dispersion has been simulated using a Computational Fluid Dynamics (CFD) approach. Possible consequences have been calculated based on concentration and dose profiles. Mild intoxication effects appear within minutes, while serious injuries and fatalities occur approximately 20 min after release. Anticipated key emergency response challenges are: (i) the time factor due to rapid onset of symptoms, (ii) the large number of casualties, and (iii) the contaminated hazard scene.

Long-term ocular consequences of sulfur mustard in seriously eye-injured war veterans.

INTRODUCTION: Sulfur mustard (SM) has been used as a dangerous chemical warfare agent since the early 20th century. Although many descriptive studies about SM-induced ocular injuries are present in the medical literature, few of them have been conducted over a large group with serious ocular involvement. MATERIALS AND METHODS: This descriptive study was conducted on 149 severe SM-intoxicated war veterans. Ocular history, anterior and posterior segment findings using a slit lamp, and direct and indirect ophthalmoscopic findings were recorded. Severity of the disease was also recorded based on a chart of the Foundation of Martyrs and Veterans Affairs. RESULTS: Ocular complains included photophobia (73.2%), sense of decreased vision (72.5%), dry eye sensation (66.4%), foreign body sensation (61.1%), tearing (46.3%), and pain (43.0%). Slit lamp findings were meibomian gland dysfunction (MGD; 96%), blepharitis, punctal closure, trichiasis, tear break-up time, and tear meniscus layer abnormality (80% to 90%). Conjunctival disturbances included vascular abnormality, ischemia, hyperemia, subconjunctival fibrosis, and pterygium. Limbal changes were abnormal vessels, limbal tissue loss and pigment loss, and pannus formation. Corneal problems included epithelial and stromal disturbances, calcium deposition, and melting. The most frequent previous surgeries were punctal closure, lamellar keratoplasty (LK), and stem cell allograft. Severity of intoxication included mild (17%), moderate (25%), and severe (57%). CONCLUSION: Chronic blepharitis and decreased tear secretion are the 2 most important and influencing factors in progression of ocular problems in SM injuries. The more severe the initial exposure, percentage of disability, and duration of ocular involvement, the higher the likelihood of mustard gas keratopathy.

The pharmaceutical industry and the German National Socialist Regime: I.G. Farben and pharmacological research.

Before the National Socialist party came to power, the German pharmaceutical industry constituted an international reference as far as the development of new medicines was concerned, having been responsible for synthetic analgesics (phenacetin, phenazones, acetylsalicylic acid), arsphenamine, barbiturates and sulfonamides. The year 1925 saw the founding of I.G. Farben (Interessen-Gemeinschaft Farbenindustrie AG), a conglomerate of companies that would monopolize the country's chemical production and come to own all its major pharmaceutical industries. During the World War II, I.G. Farben participated in numerous operations associated with the criminal activities of the Nazi executive, including the use of slave labour in plants built close to concentration camps, such as that at Auschwitz. With regard to medical and pharmacological research projects, I.G. Farben became involved in experimental programmes using patients from the Nazi regime's euthanasia programmes and healthy subjects recruited without their consent from concentration camps, on whom various pharmacological substances were tested, including sulfamide and arsenical derivatives and other preparations whose composition is not precisely known (B-1012, B-1034, 3382 or Rutenol, 3582 or Acridine), generally in relation to the treatment of infectious diseases, such as typhus, erysipelas, scarlet fever or paratyphoid diarrhoea. Furthermore, I.G. Farben played a decisive role in the German army's chemical warfare programme, contributing to the development of the first two neurotoxic substances, later known as 'nerve agents', tabun and sarin. Some of these activities came to light as a result of the one the famous Nuremberg Trials in 1947, which saw 24 executives and scientists from I.G. Farben brought to justice for, among other offences, the use of slave labour in the concentration camps and forced experimentation with drugs on prisoners.