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Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media, and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for four days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for 10 days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a calcein acetoxymethyl/ethidium homodimer-1 based live/dead staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies. [ABSTRACT FROM AUTHOR]

The exposure to highly toxic organophosphorus (OP) compounds, including pesticides and nerve agents, is an ongoing medical challenge. OP can induce the uncontrolled overstimulation of the cholinergic system through inhibition of the enzyme acetylcholinesterase (AChE). The cytochrome P450 (CYP) enzymes in the liver play a predominant role in the metabolism of xenobiotics and are involved in the oxidative biotransformation of most clinical drugs. Previous research concerning the interactions between OP and CYP has usually focused on organothiophosphate pesticides that require CYP-mediated bioactivation to their active oxon metabolites to act as inhibitors of AChE. Since there has been little data available concerning the effect of nerve agents on CYP, we performed a study with cyclosarin (GF) and O-ethyl-S-[2-(diisopropylamino)-ethyl]-methylphosphonothioate (VX) by using a well-established, metabolically competent in vitro liver model (HepaRG cells). The inhibitory effect of the nerve agents GF and VX on the CYP3A4 enzyme was investigated showing a low CYP3A4 inhibitory potency. Changes on the transcription level of CYP and associated oxygenases were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the two nerve agent concentrations 250 nM and 250 μM. In conclusion, the results demonstrated various effects on oxygenase-associated genes in dependence of the concentration and the structure of the nerve agent. Such information might be of relevance for potential interactions between nerve agents, antidotes or other clinically administered drugs, which are metabolized by the affected CYP, for example, for the therapy with benzodiazepines, that are used for the symptomatic treatment of OP poisoning and that require CYP-mediated biotransformation., (© 2024 John Wiley & Sons Ltd.)

Organophosphate pesticide poisoning challenges health care systems worldwide. Furthermore, nerve agents remain a continuous threat. The treatment options for organophosphate poisoning have virtually been unchanged for decades, relying on symptomatic treatment and the use of oximes to indirectly restore neuromuscular function. Hence, compounds targeting directly nicotinic acetylcholine receptors (nAChRs) might substantially improve treatment options. The current study investigated a series of bispyridinium analogues with a trimethylene or 2,2'-diethyloxy linker in a rat hemidiaphragm model, using indirect field stimulation. Methyl- and ethyl-substituted bispyridinium analogues restored neuromuscular function up to 37 ± 17% (MB419, a 3-methyl analogue) at a stimulation frequency of 20 Hz. The bispyridinium analogues with a 2- or 3-methyl group, or a 2- or 3-ethyl group, tended towards a higher restoration of neuromuscular function than those with a 4-methyl or 4-ethyl group, respectively. The current data can be used for future studies to optimize structure-based molecular modeling of compounds targeting the nAChR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Introduction: Intentional and unintentional organophosphorus pesticide exposure is a public health concern. Organothiophosphate compounds require metabolic bioactivation by the cytochrome P450 system to their corresponding oxon analogues to act as potent inhibitors of acetylcholinesterase. It is known that interactions between cytochrome P450 and pesticides include the inhibition of major xenobiotic metabolizing cytochrome P450 enzymes and changes on the genetic level., Methods: In this in vitro study, the influence of the pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases was investigated with a metabolically competent cell line (HepaRG cells). First, the viability of the cells after exposure to parathion and paraoxon was evaluated. The inhibitory effect of both pesticides on cytochrome P450 3A4, which is a pivotal enzyme in the metabolism of xenobiotics, was examined by determining the dose-response curve. Changes on the transcription level of 92 oxygenase associated genes, including those for important cytochrome P450 enzymes, were evaluated., Results: The exposure of HepaRG cells to parathion and paraoxon at concentrations up to 100 µM resulted in a viability of 100 per cent. After exposure for 24 hours, pronounced inhibition of cytochrome P450 3A4 enzyme activity was shown, indicating 50 per cent effective concentrations of 1.2 µM (parathion) and 2.1 µM (paraoxon). The results revealed that cytochrome P450 involved in parathion metabolism were significantly upregulated., Discussion: Relevant changes of the cytochrome P450 3A4 enzyme activity and significant alteration of genes associated with cytochrome P450 suggest an interference of pesticide exposure with numerous metabolic processes. The major limitations of the work involve the use of a single pesticide and the in vitro model as surrogate to human hepatocytes., Conclusion: The data of this study might be of relevance after survival of acute, life-threatening intoxications with organophosphorus compounds, particularly for the co-administration of drugs, which are metabolized by the affected cytochrome P450.

Organ-on-a-chip technology is considered a next-generation platform in pharmacology and toxicology. Nevertheless, this novel technology still faces several challenges concerning the respective materials which are used for these microfluidic devices. Currently available organ-chips are most often based on polydimethylsiloxane (PDMS). However, this material has strong limitations regarding compound binding. The current study investigated options to reduce compound absorption of the highly toxic nerve agent VX (1000 µmol/L) in a commercially available organ-chip. In addition, surface effects on degradation products of VX were investigated. The alternative polymer cyclic olefin copolymers (CoC) showed significantly less compound absorption compared to PDMS. Furthermore, a coating of PDMS- and CoC-based chips was investigated. The biocompatible polymer polyethyleneimine (PEI) successfully modified PDMS and CoC surfaces and further reduced compound absorption. A previously examined VX concentration after 72 h of 141 ± 10 µmol/L VX could be increased to 442 ± 54 µmol/L. Finally, the respective concentrations of VX and degradation products accounted for > 90% of the initial concentration of 1000 µmol/L VX. The currently described surface modification might be a first step towards the optimization of organ-on-a-chip surfaces, facilitating a better comparability of different studies and results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Organ-on-a-chip platforms are an emerging technology in experimental and regulatory toxicology (species-specific differences, ethical considerations). They address gaps between in vivo and in vitro models. However, there are still certain limitations considering material, setup and applicability. The current study examined the suitability of a commercially available polydimethylsiloxane-based (PDMS) organ-chip for the toxicokinetic characterization of the highly toxic nerve agent VX and the organophosphate pesticide parathion. The respective concentrations of 1000 µmol/L and 100 µmol/L VX and parathion were chosen deliberately high in order to study concentrations even if high compound absorption by PDMS might occur. Neuronal and liver spheroids, totaling 2 × 10 6 cells were used to study concentration changes of VX and parathion. In addition, VX enantiomers were quantified. The current study suggests a significant absorption of VX, respectively parathion by PDMS. This might require future investigation of alternative materials or coatings to limit absorption for organophosphorus compounds in toxicokinetic studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Dr. Franz Worek is a member of the Editorial Board of Toxicology Letters Dr. Niko Amend is Executive Guest Editor of the Special Issue in Toxicology Letters., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Isolated organ models are a versatile tool for pharmacological and toxicological research. Small bowel has been used to assess the inhibition of smooth muscle contraction by opioids. In the present study, we set out to establish a pharmacologically stimulated rat bowel model. The effects of carfentanil, remifentanil and the new synthetic opioid U-48800 and their respective antagonists naloxone, nalmefene and naltrexone were studied in a small bowel model in rats. The IC 50 values of the tested opioids were as follows: carfentanil (IC 50 = 0.02 µmol/L, CI 0.02-0.03 µmol/L) ≫ remifentanil (IC 50 = 0.51 µmol/L, CI 0.40-0.66 µmol/L) ≫ U-48800 (IC 50 = 1.36 µmol/L, CI 1.20-1.54 µmol/L). The administration of the opioid receptor antagonists naloxone, naltrexone and nalmefene led to progressive, parallel rightward shifts of the dose-response curves. Naltrexone was most potent in antagonizing the effects of U-48800, whereas naltrexone and nalmefene were most effective in antagonizing the effects of carfentanil. In summary, the current model seems to be a robust tool to study opioid effects in a small bowel model without the necessity of using electrical stimulation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Dr. Franz Worek is a member of the editorial board of Toxicology Letters. Conflict of interest statement The authors declare that there are no conflicts of interest. The study was funded by the German Ministry of Defence. However, the design, performance, data interpretation and manuscript writing were under the control of the authors and has not been influenced by the German Ministry of Defence., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Although 193 states have committed to the Chemical Weapons Convention and 98% of the declared chemical weapons stockpiles have been destroyed so far, nerve agent poisoning remains a lingering threat. The recent dissemination of sarin in Syria, the assassination of Kim Jong-Nam in Malaysia, and the assault on Sergei Skripal in the United Kingdom underline the need for effective treatment. The current therapeutic options of a muscarinic receptor antagonist, an oxime, and an anticonvulsant have been unchanged for decades. Therefore, new therapeutic strategies, for example, bioscavengers and receptor-active substances, are promising concepts that have to be examined for their benefits and limitations. In order to facilitate rapid diagnosis in challenging clinical situations, point-of-care diagnostics and detection are of importance. Therapeutic guidance concerning the duration and success of the current oxime therapy via determination of the cholinesterase status can contribute to an optimal use of resources. In summary, the challenges of current and future therapies for nerve agent poisoning and key diagnostic devices will be discussed., (© 2020 New York Academy of Sciences.)

The global use of organophosphorus compounds (OP) for pest control and nerve agents being used in military conflicts and for assassinations renders intoxications by these agents a public health concern. OP-poisoned patients often suffer from dysrhythmias which may ultimately result in death. In this study, human-induced pluripotent stem cells derived cardiomyocytes were exposed to OP compounds in a microelectrode array system (MEA). The MEA system is widely accepted to assess the proarrhythmic properties of (candidate) drugs. The directly acting cholinergic compounds acetylcholine and carbachol and the irreversible acetylcholinesterase inhibitor cyclosarin - a highly toxic nerve agent - were assessed. All three compounds induced a dose-dependent (up to 600 nmol/L) corrected field potential duration (FPDc) prolongation of 9.7 ± 0.6% for carbachol, for 9.7 ± 1.2% acetylcholine and 9.4 ± 0.5% for cyclosarin. Additionally, the electrophysiological alterations of the clinically approved oxime reactivators obidoxime, pralidoxime and the oximes in development HI-6 and MMB-4 were investigated in the absence of OP. Neither of these oximes (up to a concentration of 300 μmol/L) caused dysrhythmia nor beat arrest. The competitive muscarinic receptor antagonist atropine as a cornerstone in the treatment of OP poisoning was also analyzed. Interestingly, atropine caused a drop in the beat rate which might result from a non-receptor action of this substance in the absence of OP. Atropine in combination with the OP nerve agent cyclosarin and the direct cholinergics acetylcholine or carabachol completely reversed the induced FPDc prolongation. However, the oxime HI-6 as potent reactivator of cyclosarin-inhibited AChE was not able to prevent the FPDc prolongation in this model. In conclusion, the current model allows the assessment of FPDc prolongation by the nerve agent cyclosarin, the cholinergic compounds carbachol, acetylcholine and the block of this effect by atropine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: In a military or terrorist scenario, combination of organophosphorus compounds (OP) poisoning with physical trauma requiring surgical treatment and thus general anaesthesia are possible. Previous in vitro studies showed an altered potency of relevant anaesthetics during cholinergic crisis. Hence, it is not clear, which anaesthetics are suitable to achieve the necessary stage of surgical anaesthesia in OP poisoning., Methods: In the present study, different anaesthetic regimens (ketamine-midazolam, propofol-fentanyl, thiopental-fentanyl), relevant in military emergency medicine, were examined in soman-poisoned rats. Clinical signs and cardiovascular variables were recorded continuously. Blood samples for acetylcholinesterase (AChE) activity were drawn. After euthanasia or death of the animals, brain and diaphragm were collected for cholinesterase assays., Results: Propofol-fentanyl and thiopental-fentanyl resulted in surgical anaesthesia throughout the experiments. With ketamine-midazolam, surgical anaesthesia without respiratory impairment could not be achieved in pilot experiments (no soman challenge) and was therefore not included in the study. Soman-poisoned and control animals required a comparable amount of propofol-fentanyl or thiopental-fentanyl. In combination with atropine, significantly less propofol was needed. Survival rate was higher with thiopental compared to propofol. Atropine improved survival in both groups. Blood and tissue AChE activities were strongly inhibited after soman administration with and without atropine treatment., Discussion: The current in vivo study did not confirm concerns of altered potency of existing anaesthetic protocols for the application of propofol or thiopental with fentanyl due to soman poisoning. Despite severe cholinergic crisis, sufficient anaesthetic depth could be achieved in all animals., Conclusion: Further experiments in in vivo models closer to human pharmaco- and toxicokinetics (e.g., swine) are required for confirmation of the initial findings and for improving extrapolation to humans.

Objective: To estimate the relative risk of injury among horses deemed to be at increased risk of injury on the basis of prerace physical inspection findings and to examine the association of injury during races with race-related characteristics., Design: Cohort study., Animals: 2,187 Thoroughbred horses that started 3,227 races in Kentucky., Procedure: All race starts for which a horse was deemed to be at increased risk of injury on the basis of prerace physical inspection findings and a random sample of race starts for which horses were not deemed at increased risk of injury were included in the study. Findings of prerace physical inspection, race-related characteristics, and outcome of the race (race results and whether the horse incurred an injury) were recorded for each race start. Race starts in which a horse incurred an injury during a race were compared with race starts in which injuries were not incurred to identify factors associated with injury during races., Results: Abnormality of the suspensory ligament of the forelimbs detected during prerace physical inspection, racetrack, class of race (claiming race < or = $25,000 vs other classes), and distance of race (< 7 furlongs vs other distances) were significantly associated with increased risk of injury., Conclusions and Clinical Relevance: Prerace physical inspection findings, particularly abnormalities of the suspensory ligament, may be used to identify horses at increased risk of injury during races. Rate of injury differed among racetracks, and horses in certain types of races (lower-priced claiming races and races of shorter distance) may be at increased risk of injury during races.

Twenty-six of 39 Syrian hamsters obtained by this laboratory from a pet supplier had enteritis and showed signs of "wet tail." An enteritis was reproduced in healthy hamsters by oral inoculation of homogenized ilea and jejuna from the diseased hamsters. The most characteristic pathologicfeatures were a variably enlarged distal jejunum and ileum and granulomatous lesions in the ileal subserosal wall. Histologic findings included a greatly hyperplastic jejunal and ileal mucosa that frequently contained submucosal accumulations of inflammatory cells. Often mucosal glands extended into these areas. The gross and histologic lesions produced were identical to those described in proliferative ileitis. Bacterial cultures from infected tissues consistently grew a slow lactose-fermenting Escherichia coli. Although diarrhea could be produced by oral inoculation of the organism, the typical morphologic lesions were not produced.

Questionnaires on research activities, mortality rates observed in various age groups, extent of examination of dead hamsters, and natural disease conditions and their relative importance were returned by 24 of 43 organisations surveyed in the United States. The average preweaning mortality rate due to all causes was 11-9%. Comparative data from 6 organisations that raised 87 880 hamsters in the calendar year 1971 indicated that 97-5% of total preweaning mortality was due to cannibalism. 13-7% of all animals died before use for experiments. "Wet tail" was the most frequently recognized disease (71%), and it was also listed as the most important. Pneumonia was recognized by 43% of the respondents and was most commonly listed as second in importance. A selective review of the literature is presented on those diseases recognized by more than one survey respondent.