In Vitro Characterization and Rescue of VX Metabolism in Human Liver Microsomes.

Venomous agent X (VX) is an organophosphate acetylcholinesterase (AChE) inhibitor, and although it is one of the most toxic AChE inhibitors known, the extent of metabolism in humans is not currently well understood. The known metabolism in humans is limited to the metabolite identification from a single victim of the Osaka poisoning in 1994, which allowed for the identification of several metabolic products. VX has been reported to be metabolized in vitro by paraoxonase-1 and phosphotriesterase, although their binding constants are many orders of magnitude above the LD50, suggesting limited physiologic relevance. Using incubation with human liver microsomes (HLMs), we have now characterized the metabolism of VX and the formation of multiple metabolites as well as identified a Food and Drug Administration-approved drug [ethylenediaminetetraacetic acid (EDTA)] that enhances the metabolic rate. HLM incubation alone shows a pronounced increase in the metabolism of VX compared with buffer, suggesting that cytochrome P450-mediated metabolism of VX is occurring. We identified a biphasic decay with two distinct rates of metabolism. The enhancement of VX metabolism in multiple buffers was assessed to attempt to mitigate the effect of hydrolysis rates. The formation of VX metabolites was shown to be shifted with HLMs, suggesting a pathway enhancement over simple hydrolysis. Additionally, our investigation of hydrolysis rates in various common buffers used in biologic assays discovered dramatic differences in VX stability. The new human in vitro VX metabolic data reported points to a potential in vivo treatment strategy (EDTA) for rescue in individuals that are poisoned though enhancement of metabolism alongside existing treatments. SIGNIFICANCE STATEMENT: Venomous agent X (VX) is a potent acetylcholinesterase inhibitor and chemical weapon. To date, we do not possess a clear understanding of its metabolism in humans that would assist us in treating those exposed to it. This study now describes the human liver microsomal metabolism of VX and identifies ethylenediaminetetraacetic acid, which appears to enhance the rate of metabolism. This may provide a potential treatment option for human VX poisoning.

Evaluation of Malignant Hyperthermia Features in Patients with Pathogenic or Likely Pathogenic RYR1 Variants Disclosed through a Population Genomic Screening Program.

BACKGROUND: Malignant hyperthermia (MH) susceptibility is a heritable musculoskeletal disorder that can present as a potentially fatal hypermetabolic response to triggering anesthesia agents. Genomic screening for variants in MH-associated genes RYR1 and CACNA1S provides an opportunity to prevent morbidity and mortality. There are limited outcomes data from disclosing variants in RYR1, the most common MH susceptibility gene, in unselected populations. The authors sought to identify the rate of MH features or fulminant episodes after triggering agent exposure in an unselected population undergoing genomic screening including actionable RYR1 variants. METHODS: The MyCode Community Health Initiative by Geisinger (USA) is an electronic health record-linked biobank that discloses pathogenic and likely pathogenic variants in clinically actionable genes to patient-participants. Available electronic anesthesia and ambulatory records for participants with actionable RYR1 results returned through December 2020 were evaluated for pertinent findings via double-coded chart reviews and reconciliation. Descriptive statistics for observed phenotypes were calculated. RESULTS: One hundred fifty-two participants had an actionable RYR1 variant disclosed during the study period. None had previous documented genetic testing for MH susceptibility; one had previous contracture testing diagnosing MH susceptibility. Sixty-eight participants (44.7%) had anesthesia records documenting triggering agent exposure during at least one procedure. None received dantrolene treatment or had documented muscle rigidity, myoglobinuria, hyperkalemia, elevated creatine kinase, severe myalgia, or tea-colored urine. Of 120 possibly MH-related findings (postoperative intensive care unit admissions, hyperthermia, arterial blood gas evaluation, hypercapnia, or tachycardia), 112 (93.3%) were deemed unlikely to be MH events; 8 (6.7%) had insufficient records to determine etiology. CONCLUSIONS: Results demonstrate a low frequency of classic intraanesthetic hypermetabolic phenotypes in an unselected population with actionable RYR1 variants. Further research on the actionability of screening for MH susceptibility in unselected populations, including economic impact, predictors of MH episodes, and expanded clinical phenotypes, is necessary.

Reproductive and developmental toxicity following exposure to organophosphate ester flame retardants and plasticizers, triphenyl phosphate and isopropylated phenyl phosphate, in Sprague Dawley rats.

Two organophosphate esters used as flame retardants and plasticizers, triphenyl phosphate (TPHP) and isopropylated phenyl phosphate (IPP), have been detected in environmental samples around the world. Human exposure primarily occurs via oral ingestion with reported higher concentrations in children. Currently, there are no data to evaluate potential risk from exposure to either TPHP or IPP during fetal development. These short-term perinatal studies in rats provide preliminary toxicity data for TPHP and IPP, including information on transfer to fetus/offspring and across the pup blood-brain barrier. In separate experiments, TPHP or IPP were administered via dosed feed at concentrations 0, 1000, 3000, 10 000, 15 000, or 30 000 ppm to time-mated Hsd:Sprague Dawley SD rats from gestation day (GD) 6 through postnatal day (PND) 28; offspring were provided dosed feed at the same concentration as their dam (PND 28-PND 56). TPHP- and IPP-related toxicity resulted in removal of both 30 000 ppm groups on GD 12 and 15 000 ppm IPP group after parturition. Body weight and organ weights were impacted with exposure in remaining dams. Reproductive performance was perturbed at ≥10 000 ppm TPHP and all IPP exposure groups. In offspring, both TPHP- and IPP-related toxicity was noted in pups at ≥10 000 ppm as well as reduction in bodyweights, delays in pubertal endpoints, and/or reduced cholinesterase enzyme activity starting at 1000 ppm TPHP or IPP. Preliminary internal dose assessment indicated gestational and lactational transfer following exposure to TPHP or IPP. These findings demonstrate that offspring development is sensitive to 1000 ppm TPHP or IPP exposure.

A procedure to detect and identify specific chemicals of potential inhalation toxicity concern in aerosols.

OBJECTIVE: Understanding the potential inhalation toxicity of poorly characterized aerosols is challenging both because aerosols may contain numerous chemicals and because it is difficult to predict which chemicals may present significant inhalation toxicity concerns at the observed levels. We have developed a novel systematic procedure to address these challenges through non-targeted chemical analysis by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS) and assessment of the results using publicly available toxicity data to prioritize the tentatively identified detected chemicals according to potential inhalation toxicity. MATERIALS AND METHODS: The procedure involves non-targeted chemical analysis of aerosol samples utilizing GC × GC-TOFMS, which is selected because it is an effective technique for detecting chemicals in complex samples and assigning tentative identities according to the mass spectra. For data evaluation, existing toxicity data (e.g. from the U.S. Environmental Protection Agency CompTox Chemicals Dashboard) are used to calculate multiple toxicity metrics that can be compared among the tentatively identified chemicals. These metrics include hazard quotient, incremental lifetime cancer risk, and metrics analogous to hazard quotient that we designated as exposure-(toxicology endpoint) ratios. RESULTS AND DISCUSSION: We demonstrated the utility of our procedure by detecting, identifying, and prioritizing specific chemicals of potential inhalation toxicity concern in the mainstream smoke generated from the machine-smoking of marijuana blunts. CONCLUSION: By designing a systematic approach for detecting and identifying numerous chemicals in complex aerosol samples and prioritizing the chemicals in relation to different inhalation toxicology endpoints, we have developed an effective approach to elucidate the potential inhalation toxicity of aerosols.

A Novel, Modified Human Butyrylcholinesterase Catalytically Degrades the Chemical Warfare Nerve Agent, Sarin.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282-285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.

Characterization of Cholinesterases From Multiple Large Animal Species for Medical Countermeasure Development Against Chemical Warfare Nerve Agents.

Organophosphorus (OP) compounds, which include insecticides and chemical warfare nerve agents (CWNAs) such as sarin (GB) and VX, continue to be a global threat to both civilian and military populations. It is widely accepted that cholinesterase inhibition is the primary mechanism for acute OP toxicity. Disruption of cholinergic function through the inhibition of acetylcholinesterase (AChE) leads to the accumulation of the neurotransmitter acetylcholine. Excess acetylcholine at the synapse results in an overstimulation of cholinergic neurons which manifests in the common signs and symptoms of OP intoxication (miosis, increased secretions, seizures, convulsions, and respiratory failure). The primary therapeutic strategy employed in the United States to treat OP intoxication includes reactivation of inhibited AChE with the oxime pralidoxime (2-PAM) along with the muscarinic acetylcholine receptor antagonist atropine and the benzodiazepine, diazepam. CWNAs are also known to inhibit butyrylcholinesterase (BChE) without any apparent toxic effects. Therefore, BChE may be viewed as a "bioscavenger" that stoichiometrically binds CWNAs and removes them from circulation. The degree of inhibition of AChE and BChE and the effectiveness of 2-PAM are known to vary among species. Animal models are imperative for evaluating the efficacy of CWNA medical countermeasures, and a thorough characterization of available animal models is important for translating results to humans. Thus, the objective of this study was to compare the circulating levels of each of the cholinesterases as well as multiple kinetic properties (inhibition, reactivation, and aging rates) of both AChE and BChE derived from humans to AChE and BChE derived from commonly used large animal models.

Kinetic analysis of oxime-assisted reactivation of human, Guinea pig, and rat acetylcholinesterase inhibited by the organophosphorus pesticide metabolite phorate oxon (PHO).

Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl2, MMB4-DMS, and HLö7 DMS. The inhibition rate constants (ki) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8×103 to 1.4×104M-1min-1) compared to many other OPs. Obidoxime Cl2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48h) for all three species. CONCLUSIONS: (1) Obidoxime Cl2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.

Efficacy of Recommended Prehospital Human Equivalent Doses of Atropine and Pralidoxime Against the Toxic Effects of Carbamate Poisoning in the Hartley Guinea Pig.

PURPOSE: Aldicarb and methomyl are carbamate pesticides commonly implicated in human poisonings. The primary toxic mechanism of action for carbamate poisoning is cholinesterase (ChE) inhibition. As such, it is logical to assume that the currently accepted therapies for organophosphate poisoning (muscarinic antagonist atropine and the oxime acetylcholinesterase reactivator pralidoxime chloride [2-PAM Cl]) could afford therapeutic protection. However, oximes have been shown to be contraindicated for poisoning by some carbamates. METHODS: A protective ratio study was conducted in guinea pigs to evaluate the efficacy of atropine and 2-PAM Cl. The ChE activity was determined in both the blood and the cerebral cortex. RESULTS: Coadministration of atropine free base (0.4 mg/kg) and 2-PAM Cl (25.7 mg/kg) demonstrated protective ratios of 2 and 3 against aldicarb and methomyl, respectively, relative to saline. The data reported here show that this protection was primarily mediated by the action of atropine. The reactivator 2-PAM Cl had neither positive nor negative effects on survival. Both blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were significantly reduced at 15 minutes postchallenge but gradually returned to normal within 24 hours. Analysis of cerebral cortex showed that BChE, but not AChE, activity was reduced in animals that succumbed prior to 24 hours after challenge. CONCLUSION: The results suggest that coadministration of atropine and 2-PAM Cl at the currently recommended human equivalent doses for use in the prehospital setting to treat organophosphorus nerve agent and pesticide poisoning would likely also be effective against aldicarb or methomyl poisoning.

The Pesticide Metabolites Paraoxon and Malaoxon Induce Cellular Death by Different Mechanisms in Cultured Human Pulmonary Cells.

Organophosphorus (OP) pesticides are known to induce pulmonary toxicity in both humans and experimental animals. To elucidate the mechanism of OP-induced cytotoxicity, we examined the effects of parathion and malathion and their respective metabolites, paraoxon and malaoxon, on primary cultured human large and small airway cells. Exposure to paraoxon and malaoxon produced a dose-dependent increase in cytotoxicity following a 24-hour exposure, while treatment with parathion or malathion produced no effects at clinically relevant concentrations. Exposure to paraoxon-induced caspase activation, but malaoxon failed to induce this response. Since caspases have a major role in the regulation of apoptosis and cell death, we evaluated OP-induced cell death in the presence of a caspase inhibitor. Pharmacological caspase inhibition protected against paraoxon-induced cell death but not malaoxon-induced cell death. These data suggest that caspase activation is a key signaling element in paraoxon-induced cell death, but not malaoxon-induced cellular death in the pulmonary epithelium.

Chemical warfare agent and biological toxin-induced pulmonary toxicity: could stem cells provide potential therapies?

Chemical warfare agents (CWAs) as well as biological toxins present a significant inhalation injury risk to both deployed warfighters and civilian targets of terrorist attacks. Inhalation of many CWAs and biological toxins can induce severe pulmonary toxicity leading to the development of acute lung injury (ALI) as well as acute respiratory distress syndrome (ARDS). The therapeutic options currently used to treat these conditions are very limited and mortality rates remain high. Recent evidence suggests that human stem cells may provide significant therapeutic options for ALI and ARDS in the near future. The threat posed by CWAs and biological toxins for both civilian populations and military personnel is growing, thus understanding the mechanisms of toxicity and potential therapies is critical. This review will outline the pulmonary toxic effects of some of the most common CWAs and biological toxins as well as the potential role of stem cells in treating these types of toxic lung injuries.

Evaluation of HemogloBind™ treatment for preparation of samples for cholinesterase analysis.

Acetylcholine is an essential neurotransmitter found throughout the nervous system. Its action on postsynaptic receptors is regulated through hydrolysis by various carboxylesterases, especially cholinesterases (ChEs). The acute toxicity of organophosphate (OP) compounds is directly linked to their action as inhibitors of ChE. One widely used assay for evaluating ChE activity is a spectrophotometric method developed by Ellman et al. When the enzyme source is from tissues or, in particular, blood, hemoglobin displays a spectrophotometric peak at the same wavelength used to analyze cholinergic activity. This creates a substantial background that interferes with the Ellman's assay and must be overcome in order to accurately monitor cholinesterase activity. Herein, we directly compare blood processing methods: classical method (1.67 ± 0.30 U/mL) and HemogloBind™ treatment (1.51 ± 0.17 U/mL), and clearly demonstrate that pretreatment of blood samples with Hemoglobind™ is both a sufficient and rapid sample preparation method for the assessment of ChE activity using the Ellman's method.

Intronic polymorphisms affecting alternative splicing of human dopamine D2 receptor are associated with cocaine abuse.

The dopamine receptor D2 (encoded by DRD2) is implicated in susceptibility to mental disorders and cocaine abuse, but mechanisms responsible for this relationship remain uncertain. DRD2 mRNA exists in two main splice isoforms with distinct functions: D2 long (D2L) and D2 short (D2S, lacking exon 6), expressed mainly postsynaptically and presynaptically, respectively. Two intronic single-nucleotide polymorphisms (SNPs rs2283265 (intron 5) and rs1076560 (intron 6)) in high linkage disequilibrium (LD) with each other have been reported to alter D2S/D2L splicing and several behavioral traits in human subjects, such as memory processing. To assess the role of DRD2 variants in cocaine abuse, we measured levels of D2S and D2L mRNA in human brain autopsy tissues (prefrontal cortex and putamen) obtained from cocaine abusers and controls, and genotyped a panel of DRD2 SNPs (119 abusers and 95 controls). Robust effects of rs2283265 and rs1076560 on reducing formation of D2S relative to D2L were confirmed. The minor alleles of rs2283265/rs1076560 were considerably more frequent in Caucasians (18%) compared with African Americans (7%). Also, in Caucasians, rs2283265/rs1076560 minor alleles were significantly overrepresented in cocaine abusers compared with controls (rs2283265: 25 to 9%, respectively; p=0.001; OR=3.4 (1.7-7.1)). Several SNPs previously implicated in diverse clinical association studies are in high LD with rs2283265/rs1076560 and could have served as surrogate markers. Our results confirm the role of rs2283265/rs1076560 in D2 alternative splicing and support a strong role in susceptibility to cocaine abuse.

Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta-arrestin 2-dependent MAPK-mediated pathways.

GTP binding regulatory protein (G protein)-coupled receptors can activate MAPK pathways via G protein-dependent and -independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and beta-arrestin 2 pathways in kappa opioid receptor-induced, extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non-nitrogenous agonist, C(2)-methoxymethyl salvinorin B (MOM-Sal-B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gbetagamma subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of beta-arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM-Sal-B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM-Sal-B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gbetagamma subunits or beta-arrestin 2, suggesting that both G protein-dependent and -independent ERK pathways are required for this outcome.

Nxf and Fbxo33: novel seizure-responsive genes in mice.

Much is understood about the response of the brain to seizure but little is known in relation to the underlying molecular mechanisms involved. We used microarray technology to investigate the complex genetic response of the brain to generalized seizure. For this investigation a seizure-specific mouse brain cDNA library was generated and spotted onto microarray slides with the aim of increasing the likelihood of identifying novel genes responsive to seizure. Microarray analysis was performed on mouse hippocampus 1 h after generalized seizure pharmacologically induced by pentylenetetrazol (PTZ). Using the custom microarray slides, six genes were identified as being up-regulated in this seizure model and results were validated by real-time PCR. Four of the seizure-responsive genes had previously-reported roles in apoptosis, proliferation or differentiation of neural cells. Two of the genes were novel and in situ hybridization analysis demonstrated heightened mRNA expression in the hippocampus 1 h following generalized convulsive seizure, in a pattern which is typical for other activity-dependant genes expressed in this structure. In addition to being up-regulated postseizure, the genes described in this paper appear to be expressed normally in the adult hippocampus and during development.

Acute gout precipitated by total parenteral nutrition.

Hypouricemia is seen in a variety of clinical situations. Although precipitation of gout is well known following initiation of uricosuric therapy, it has been reported rarely following the institution of total parenteral nutrition (TPN), despite its known uricosuric effect. We describe a patient who developed polyarticular gout on 2 occasions after a sudden decline in serum uric acid after initiation of purine-free TPN. Potential etiologies include increased urate clearance due to the infusion of glycine or amino acids. Monitoring of serum uric acid concentrations in patients with a history of gout may help predict a gout attack. Prophylactic treatment or alternative TPN formulations may be indicated.

Melatonin and activity rhythm responses to light pulses in mice with the Clock mutation.

Melatonin and wheel-running rhythmicity and the effects of acute and chronic light pulses on these rhythms were studied in Clock(Delta19) mutant mice selectively bred to synthesize melatonin. Homozygous melatonin-proficient Clock(Delta19) mutant mice (Clock(Delta19/Delta19)-MEL) produced melatonin rhythmically, with peak production 2 h later than the wild-type controls (i.e., just before lights on). By contrast, the time of onset of wheel-running activity occurred within a 20-min period around lights off, irrespective of the genotype. Melatonin production in the mutants spontaneously decreased within 1 h of the expected time of lights on. On placement of the mice in continuous darkness, the melatonin rhythm persisted, and the peak occurred 2 h later in each cycle over the first two cycles, consistent with the endogenous period of the mutant. This contrasted with the onset of wheel-running activity, which did not shift for several days in constant darkness. A light pulse around the time of expected lights on followed by constant darkness reduced the expected 2-h delay of the melatonin peak of the mutants to approximately 1 h and advanced the time of the melatonin peak in the wild-type mice. When the Clock(Delta19/Delta19)-MEL mice were maintained in a skeleton photoperiod of daily 15-min light pulses, a higher proportion entrained to the schedule (57%) than melatonin-deficient mutants (9%). These results provide compelling evidence that mice with the Clock(Delta19) mutation express essentially normal rhythmicity, albeit with an underlying endogenous period of 26-27 h, and they can be entrained by brief exposure to light. They also raise important questions about the role of Clock in rhythmicity and the usefulness of monitoring behavioral rhythms compared with hormonal rhythms.