Transcriptomic Characterization of Inhalation Phosphine Toxicity in Adult Male Sprague-Dawley Rats.

Phosphine (PH3) is a highly toxic, corrosive, flammable, heavier-than-air gas that is a commonly used fumigant. When used as a fumigant, PH3 can be released from compressed gas tanks or produced from commercially available metal phosphide tablets. Although the mechanism of toxicity is unclear, PH3 is thought to be a metabolic poison. PH3 exposure induces multiorgan toxicity, and no effective antidotes or therapeutics have been identified. Current medical treatment consists largely of supportive care and maintenance of cardiovascular function. To better characterize the mechanism(s) driving PH3-induced toxicity, we have performed transcriptomic analysis on conscious adult male Sprague-Dawley rats following whole-body inhalation exposure to phosphine gas at various concentration-time products. PH3 exposure induced concentration- and time-dependent changes in gene expression across multiple tissues. These gene expression changes were mapped to pathophysiological responses using molecular pathway analysis. Toxicity pathways indicative of cardiac dysfunction, cardiac arteriopathy, and cardiac enlargement were identified. These cardiotoxic responses were linked to apelin-mediated cardiomyocyte and cardiac fibroblast signaling pathways. Evaluation of gene expression changes in blood revealed alterations in pathways associated with the uptake, transport, and utilization of iron. Altered erythropoietin signaling was also observed in the blood. Upstream regulator analysis identified several therapeutics predicted to counteract PH3-induced gene expression changes. These include antihypertensive drugs (losartan, candesartan, and prazosin) and therapeutics to reduce pathological cardiac remodeling (curcumin and TIMP3). This transcriptomics study has characterized molecular pathways involved in PH3-induced cardiotoxicity. These data will aid in elucidating a precise mechanism of toxicity for PH3 and guide the development of effective medical countermeasures for PH3-induced toxicity.

Cyanide Poisoning Compromises Gene Pathways Modulating Cardiac Injury in Vivo.

Smoke inhalation from a structure fire is a common route of cyanide poisoning in the U.S. Cyanide inhibits cellular respiration, often leading to death. Its rapid distribution throughout the body can result in injuries to multiple organs, and cyanide victims were reported to experience myocardial infarction and other cardiac complications. However, molecular mechanisms of such complications are yet to be elucidated. While FDA-approved CN antidotes such as sodium thiosulfate and hydroxocobalamin are clinically used, they have foreseeable limitations during mass casualty situations because they require intravenous administration. To facilitate the development of better antidotes and therapeutic treatments, a global view of molecular changes induced by cyanide exposure is necessary. As an exploratory pursuit, we performed oligonucleotide microarrays to establish cardiac transcriptomes of an animal model of nose-only inhalation exposure to hydrogen cyanide (HCN), which is relevant to smoke inhalation. We also profiled cardiac transcriptomes after subcutaneous injection of potassium cyanide (KCN). Although the KCN injection model has often been used to evaluate medical countermeasures, this study demonstrated that cardiac transcriptomes are largely different from that of the HCN inhalation model at multiple time points within 24 h after exposure. Pathway analysis identified that HCN-induced transcriptomes were enriched with genes encoding mediators of pathways critical in modulation of cardiac complications and that a large number of such genes were significantly decreased in expression. We utilized the upstream regulatory analysis to propose drugs that can be potentially employed to treat cyanide-induced cardiac complications.

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.

Evaluating mice lacking serum carboxylesterase as a behavioral model for nerve agent intoxication.

Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.

Conceptual approaches for treatment of phosgene inhalation-induced lung injury.

Toxic industrial chemicals are used throughout the world to produce everyday products such as household and commercial cleaners, disinfectants, pesticides, pharmaceuticals, plastics, paper, and fertilizers. These chemicals are produced, stored, and transported in large quantities, which poses a threat to the local civilian population in cases of accidental or intentional release. Several of these chemicals have no known medical countermeasures for their toxic effects. Phosgene is a highly toxic industrial chemical which was used as a chemical warfare agent in WWI. Exposure to phosgene causes latent, non-cardiogenic pulmonary edema which can result in respiratory failure and death. The mechanisms of phosgene-induced pulmonary injury are not fully identified, and currently there is no efficacious countermeasure. Here, we provide a proposed mechanism of phosgene-induced lung injury based on the literature and from studies conducted in our lab, as well as provide results from studies designed to evaluate survival efficacy of potential therapies following whole-body phosgene exposure in mice. Several therapies were able to significantly increase 24h survival following an LCt50-70 exposure to phosgene; however, no treatment was able to fully protect against phosgene-induced mortality. These studies provide evidence that mortality following phosgene toxicity can be mitigated by neuro- and calcium-regulators, antioxidants, phosphodiesterase and endothelin receptor antagonists, angiotensin converting enzymes, and transient receptor potential cation channel inhibitors. However, because the mechanism of phosgene toxicity is multifaceted, we conclude that a single therapeutic is unlikely to be sufficient to ameliorate the multitude of direct and secondary toxic effects caused by phosgene inhalation.

A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity.

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.

Mammalian electron transferring flavoprotein.flavoprotein dehydrogenase complexes observed by microelectrospray ionization-mass spectrometry and surface plasmon resonance.

Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein.flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein.electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase.electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 microm for the dimethylglycine dehydrogenase-electron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.