Abstract
Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25–16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.
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Abbreviations
- MMB4:
-
1,1-Methylene bis[4(hydroxyimino) methyl]pyridinium
- 2-PAM:
-
2-Pyridine aldoxime
- AChE:
-
Acetylcholinesterase
- Ach:
-
Acetylcholine
- BTX:
-
α-Bungarotoxin
- AUC:
-
Area under-the-curve
- EPP:
-
Endplate potential
- EDL:
-
Extensor digitorum longus
- FDB:
-
Flexor digitorum brevis
- IC50:
-
Medial inhibitory concentration
- LD50:
-
Median lethal dose
- mEPP:
-
Miniature endplate potentials
- CTX:
-
µ-Conotoxin GIIIB
- NMJ:
-
Neuromuscular junction
- OP:
-
Organophosphate
- QC:
-
Quantal content
- RMP:
-
Resting membrane potential
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Acknowledgements
We would like to acknowledge technical assistance by Marian Nelson, Megan Lyman, Celinia Ondeck, Kyle Kelly, Mark Mangkhalakhili and Dr. Edwin Vazquez-Cintron. This work was conducted under funding from the Defense Threat Reduction Agency–Joint Science and Technology Office, Medical S&T Division (project CB3945). This research was performed while JM, SW, KP and KB held Oak Ridge Institute of Science and Engineering Fellowship awards and BW held a Defense Threat Reduction Agency-National Research Council Research Associateship award. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.
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204_2020_2858_MOESM1_ESM.eps
Supplementary file1 Supplemental Figure 1. Summary of experimental approach for muscle function assays. (A) Representative peak amplitude plot from a rat control experiment demonstrating how muscle tensions were analyzed over time. Phrenic nerve-hemidiaphragm preparations were stimulated at suprathreshold voltages (rat: 5 mV; rabbit: 10 mV) in 10 min iterations involving 29 twitch impulses (0.05 Hz; low-amplitude spikes) followed by a tetanic stimulation at half-maximal force frequency (rat: 25 impulses at 40 Hz; rabbit: 50 impulses at 42.5 Hz; higher amplitude spike). The amplitudes of the last five twitch tensions (orange) were averaged, and the area under the curve (AUC) of the tetanic tensions (blue) was measured to produce a twitch and tetanic value for each iteration. Each experiment lasted a total of 120 min, involving a 30 min control period to confirm the stability of the preparation and a 90 min post-treatment period. Twitch tensions and tetanic AUCs were normalized to average control values. (B-G) Representative rat traces (top panels) and rabbit traces (bottom panels) demonstrating concentration- and time-dependent effects of MMB4 on nerve-elicited twitches and tetanic tensions. The effects of MMB4 on twitch amplitudes and tetanic AUCs were determined during each interval, normalized to mean control values and compared to vehicle-treated hemidiaphragms. Individual spikes are not discernable because of the time base necessary to demonstrate 120 min of data. (H, I) Rat (top, n = 3 each) and rabbit (bottom, n = 3 each) hemidiaphragms were treated with vehicle (black) or 16 mM MMB4 (red) at time 0 (first arrow) and monitored for changes in twitch tensions at 10 min intervals. Once twitch tensions stabilized, MMB4 was removed by three full media changes (second arrow), and hemidiaphragms were monitored for recovery of twitch tensions through 90 min (EPS 5700 kb)
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Supplementary file2 Supplemental Figure 2. Concentration-dependent effects of nicotine and acetylcholine on postsynaptic currents in mouse flexor digitorum brevis (FDB) muscle fibers. Dissociated FDB muscle fibers were superfused with increasing concentrations of agonist, and the resulting endplate currents were recorded by the whole-cell patch clamp technique. (A) Consistent with previous reports, nicotine superfusion activated inward currents with increasing amplitudes that peaked at 300–600 µM and sharply declined at higher concentrations (Jadey et al. 2013). (B) ACh superfusion produces a monotonic increase in endplate currents up to the highest concentration tested (1000 µM). Further details regarding sample sizes and treatment conditions are provided in Supplemental Table 1 (EPS 470 kb)
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Supplementary file3 Supplemental Figure 3. Frequency-dependent effects of MMB4 on EPP generation in rat and rabbit phrenic-nerve hemidiaphragm preparations. Average waveforms in response to twenty EPPs elicited from rat and rabbit diaphragm endplates at 12.5 Hz, 25 Hz, 50 Hz, and 100 Hz before and 30 min after the addition of 8 mM MMB4 (n = 4 each). (A-H) Representative EPPs from naïve endplates (black) and rat (cyan; A-D) or rabbit endplates (purple; E-H) treated with 8 mM MMB4 (EPS 1835 kb)
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Supplementary file4 Supplemental Figure 4. Frequency-dependent effects of MMB4 on EPP generation in rat and rabbit phrenic-nerve hemidiaphragm preparations. Average depolarization in response to twenty EPPs elicited from rat and rabbit diaphragm endplates at 12.5 Hz, 25 Hz, 50 Hz, and 100 Hz before and 30 min after the addition of 8 mM MMB4 (n = 4 each). (A-H) EPP amplitudes during high frequency stimulation at the indicated frequencies in rat (C-F) and rabbit (G-J) hemidiaphragms. Consistent with single stimulus studies in Figure 2, 8 mM MMB4 depressed EPP amplitudes. However, stimulation trains reveal differences in EPP rundown between vehicle-treated (black) and MMB4-treated (cyan or purple) hemidiaphragms. Further details regarding statistical tests and sample sizes are provided in Supplemental Table 1 (EPS 928 kb)
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Supplementary file5 Supplemental Table 1. Summary of statistical comparisons, conditions and sample sizes for all studies, including definition of individual data points and data normalization methods (PDF 77 kb)
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Machamer, J.B., Apland, J.P., Winner, B.M. et al. Functional basis for dose-dependent antagonism of rat and rabbit neuromuscular transmission by the bis-pyridinium oxime MMB4. Arch Toxicol 94, 3877–3891 (2020). https://doi.org/10.1007/s00204-020-02858-4
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DOI: https://doi.org/10.1007/s00204-020-02858-4