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.

The physiology and toxicology of acute inhalation phosphine poisoning in conscious male rats.

Phosphine (PH3) is a toxidrome-spanning chemical that is widely used as an insecticide and rodenticide. Exposure to PH3 causes a host of target organ and systemic effects, including oxidative stress, cardiopulmonary toxicity, seizure-like activity and overall metabolic disturbance. A custom dynamic inhalation gas exposure system was designed for the whole-body exposure of conscious male Sprague-Dawley rats (250-350 g) to PH3. An integrated plethysmography system was used to collect respiratory parameters in real-time before, during and after PH3 exposure. At several time points post-exposure, rats were euthanized, and various organs were removed and analyzed to assess organ and systemic effects. The 24 h post-exposure LCt50, determined by probit analysis, was 23,270 ppm × min (32,345 mg × min/m3). PH3 exposure affects both pulmonary and cardiac function. Unlike typical pulmonary toxicants, PH3 induced net increases in respiration during exposure. Gross observations of the heart and lungs of exposed rats suggested pulmonary and cardiac tissue damage, but histopathological examination showed little to no observable pathologic changes in those organs. Gene expression studies indicated alterations in inflammatory processes, metabolic function and cell signaling, with particular focus in cardiac tissue. Transmission electron microscopy examination of cardiac tissue revealed ultrastructural damage to both tissue and mitochondria. Altogether, these data reveal that in untreated, un-anesthetized rats, PH3 inhalation induces acute cardiorespiratory toxicity and injury, leading to death and that it is characterized by a steep dose-response curve. Continued use of our interdisciplinary approach will permit more effective identification of therapeutic windows and development of rational medical countermeasures and countermeasure strategies.

Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast.

BACKGROUND: The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/CAma1. In this study we investigated how Ama1p is down regulated at the completion of meiosis. FINDINGS: Here we show that Ama1p is a substrate of APC/CCdc20 but not APC/CCdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/CCdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a "KEN-box" like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/CAma1-mediated Cdc20p degradation. CONCLUSIONS: Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.

RXP-E: a connexin43-binding peptide that prevents action potential propagation block.

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing approximately 20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH(i) 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.

Identification of a novel peptide that interferes with the chemical regulation of connexin43.

The carboxyl-terminal domain of connexin43 (Cx43CT) is involved in various intra- and intermolecular interactions that regulate gap junctions. Here, we used phage display to identify novel peptidic sequences that bind Cx43CT and modify Cx43 regulation. We found that Cx43CT binds preferentially to peptides containing a sequence RXP, where X represents any amino acid and R and P correspond to the amino acids arginine and proline, respectively. A biased "RXP library" led to the identification of a peptide (dubbed "RXP-E") that bound Cx43CT with high affinity. Nuclear magnetic resonance data showed RXP-E-induced shifts in the resonance peaks of residues 343 to 346 and 376 to 379 of Cx43CT. Patch-clamp studies revealed that RXP-E partially prevented octanol-induced and acidification-induced uncoupling in Cx43-expressing cells. Moreover, RXP-E increased mean open time of Cx43 channels. The full effect of RXP-E was dependent on the integrity of the CT domain. These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias.