Paraoxon-Induced Protein Expression Changes to SH-SY5Y Cells

SH-SY5Y neuroblastoma cells were examined to determine changes in protein expression following exposure to the organophosphate paraoxon (O,O-diethyl-p-nitrophenoxy phosphate). Exposure of SH-SY5Y cells to paraoxon (20 μM) for 48 h showed no significant change in cell viability as established using an MTT assay. Protein expression changes from the paraoxon-treated SH-SY5Y cells were determined using a comparative, subproteome approach by fractionation into cytosolic, membrane, nuclear, and cytoskeletal fractions. The fractionated proteins were separated by 2D-PAGE, identified by MALDI-TOF mass spectrometry, and expression changes determined by densitometry. Over 400 proteins were separated from the four fractions, and 16 proteins were identified with altered expression ≥1.3-fold including heat shock protein 90 (−1.3-fold), heterogeneous nuclear ribonucleoprotein C (+2.8-fold), and H+ transporting ATP synthase beta chain (−3.1-fold). Western blot analysis conducted on total protein isolates confirmed the expression changes in these three proteins

Thionate versus Oxon: Comparison of Stability, Uptake, and Cell Toxicity of (¹⁴CH₃O)₂-Labeled Methyl Parathion and Methyl Paraoxon with SH-SY5Y Cells

The stability, hydrolysis, and uptake of the organophosphates methyl parathion and methyl paraoxon were investigated in SH-SY5Y cells. The stabilities of (14CH3O)2-methyl parathion (14C-MPS) and (14CH3O)2-methyl paraoxon (14C-MPO) at 1 μM in culture media had similar half-lives of 91.7 and 101.9 h, respectively. However, 100 μM MPO caused >95% cytotoxicity at 24 h, whereas 100 μM MPS caused 4−5% cytotoxicity at 24 h (∼60% cytotoxicity at 48 h). Greater radioactivity was detected inside cells treated with MPO as compared to MPS, although >80% of the total MPO uptake was primarily dimethyl phosphate (DMP). Maximum uptake was reached after 48 h of 14C-MPS or 14C-MPO exposure with total uptakes of 1.19 and 1.76 nM/106 cells for MPS and MPO, respectively. The amounts of MPS and MPO detected in the cytosol after 48 h of exposure time were 0.54 and 0.37 nM/106 cells, respectively

Stereoselective Addition of Dimethyl Thiophosphite to Imines

Dimethyl thiophosphite (DMTP) was synthesized from dimethyl phosphite, and the diastereoselective addition of DMTP to benzaldimines bearing chiral auxiliary groups was examined. Yields of the product α-aminophosphonothionates ranged from 17% to 75% after chromatography. The addition of DMTP to the benzaldimine derived from (S)-phenylglycinol afforded the highest diastereoselectivity (83:17), whereas addition of DMTP to the benzaldimine derived from threonine methyl ester and alanine methyl ester were far less diastereoselective, affording 38:62 and 61:39 ratios, respectively. Addition of DMTP to the benzaldimine derived from (R)-α-methylbenzylamine (78:22) and (S)-serine methyl ester (73:27) were intermediate in selectivity. DMTP addition to the imines formed between serine methyl ester and acetaldehyde and isobutyraldehyde gave 55:45 and 70:30 ratios, respectively, with the diastereoselectivity corresponding roughly to the size of the α-alkyl group. The stereochemistry of the newly formed α-stereocenters resulting from the addition of DMTP to (S)- and (R)-phenylglycinol benzaldimines was confirmed by conversion of the product α-aminophosphonothionates to the known enantiomers of phosphonophenylglycine

The organophosphorus fluorophosphonate (FP) probe does not phosphorylate M2 muscarinic receptors in guinea pig heart.

<p>Guinea pig heart cell membrane preparations with abundant expression of M2 muscarinic receptors or BSA were reacted with a fluorophosphonate tethered to a biotin group (FP-biotin) for 24 hr prior to separation by acrylamide gel electrophoresis. Blots of these gels were probed with streptavidin tagged with an infrared fluorophore (A, red in the merged image in panel C) to localize the biotin tag and with anti-M2 receptor antibody conjugated to a different infrared fluorophore (B, green in the merged image in panel C). As indicated in the merged image (C), proteins in the heart membranes that were biotinylated by the FP probe (red) did not co-localize with bands recognized by the anti-M2 receptor antibodies (green).</p

Acute intravenous administration of parathion or paraoxon did not significantly potentiate bradycardia in guinea pigs.

<p>Bradycardia was measured in anesthetized guinea pigs in response to electrical stimulation of both vagus nerves or to intravenous ACh before and after intravenous administration of parathion (1 mg/kg) or paraoxon (100 ng/kg or 100 µg/kg). None of the OP treatments had any effect on vagally-induced bradycardia at any of the frequencies tested (A). Baseline bradycardia at 5, 10, and 15 Hz was 37.62±4.13, 103.89±33.15, and 155.95±30.16 beats per minute before 100 ng/kg paraoxon administration, 47.78±22.82, 132.94±12.43, and 218.33±9.62 beats per minute before 100 µg/kg paraoxon administration, and 41.67±4.41, 85.00±7.64, and 203.33±6.67 beats per minute before 1.0 mg/kg parathion administration. (B) The higher dose of paraoxon (100 µg/kg) slightly but not significantly potentiated ACh-induced bradycardia. Data are presented as mean ± SE; n = 4–7 guinea pigs.</p

Effects of acute intravenous administration of parathion or paraoxon on bronchoconstriction in guinea pigs.

<p>Bronchoconstriction was measured in anesthetized guinea pigs in response to electrical stimulation of both vagus nerves or to intravenous ACh before and after intravenous administration of parathion (1 mg/kg) or paraoxon (100 ng/kg or 100 µg/kg). Baseline bronchoconstrictions were within normal physiological parameters (8–17 mmH2O at 5 Hz, 17–59 mmH2O at 10 Hz, and 51–127 mmH2O at 15 Hz). Parathion (hatched bars) inhibited vagally-induced bronchoconstriction by approximately 50% at all three frequencies (A). In contrast, paraoxon at 100 ng/kg (gray bars) and 100 µg/kg (black bars) acutely increased vagally-induced bronchoconstriction in a frequency-dependent manner (A). Paraoxon at 100 ng/kg did not potentiate ACh-induced bronchoconstriction (B) or significantly inhibit AChE activity (C). However, at 100 µg/kg, paraoxon potentiated ACh-induced bronchoconstriction (B) and inhibited AChE (C). Data are presented as mean ± SE; n = 3–4 guinea pigs (* <i>p</i><0.05).</p

Paraoxon does not alter expression of M2 muscarinic receptor protein.

<p>Rat sympathetic neurons in cell culture (A) or Cos-7 cells transfected with full length cDNA encoding human M2 receptor (B) were treated with paraoxon (1–1000 nM) or vehicle (0.1% DMSO) for 24 hr in the absence or presence of carbachol (1 mM). Muscarinic receptor expression was determined as specific binding of [³H]-NMS (1 nM; surface receptors) or [³H]-QNB (1 nM; total receptors) in the absence (total) or presence (non-specific binding) of atropine (0.1 M). In both cell types, carbachol significantly decreased [³H]-NMS (A and B, open bars), but not [³H]-QNB (C, open bars) binding. In both sympathetic neurons (A) and Cos-7 cells (B), paraoxon had no effect on [³H]-NMS or [³H]-QNB binding in the absence or presence of carbachol. Data are represented as mean ± SE; n = 3–5.</p

Neuronal M2 muscarinic receptor mRNA expression is not altered by paraoxon.

<p>(A) M2 receptor transcript levels in human SN-N-SH cells were not changed by exposure to paraoxon (0.1–1000 nM) for either 4 hr (gray bars) or 24 hr (black bars), while TNFα (2 ng/ml, 4 hour exposure, white bar) significantly suppressed M2 expression. (B) Exposure for 24 hr to a similar range of paraoxon concentrations had no effect on M2 mRNA levels in rat sympathetic neurons. Data are expressed as a % of control levels (cultures exposed to 0.1% DMSO) and are presented as mean ± SE; n = 3 independent cultures per treatment group (*p<0.05).</p

Paraoxon potentiates electrical field stimulation (EFS)-induced contractions in isolated guinea pig trachea and ileum.

<p>Contractions in response to EFS (10 Hz, 100 V, 0.2 ms, 5 s duration every 30 s) and ACh (5 µM) were measured in trachea (A) and ileum (B) before and after addition of paraoxon or vehicle (DMSO, 0.1% final). EFS induced contraction was measured for 30 min after drug treatment, while ACh-induced contractions were measured 35 min after drug treatment. (A) Paraoxon potentiated EFS-induced contractions in the trachea with significant effects at 360 nM; paraoxon at 100 and 360 nM significantly potentiated ACh-induced contractions. (B) In contrast, paraoxon did not significantly potentiate either EFS-induced or ACh-induced contractions in the ileum even at concentrations that significantly inhibited AChE activity. (C) Concentrations of physostigmine that significantly inhibited AChE activity to the same degree as paraoxon did not potentiate EFS-induced contractions in the ileum. Data are presented as mean ± SE; n = 4–6 guinea pigs (*<i>p</i><0.05).</p

A Novel Fluorine-18 β‑Fluoroethoxy Organophosphate Positron Emission Tomography Imaging Tracer Targeted to Central Nervous System Acetylcholinesterase

Radiosynthesis of a fluorine-18 labeled organophosphate (OP) inhibitor of acetylcholinesterase (AChE) and subsequent positron emission tomography (PET) imaging using the tracer in the rat central nervous system are reported. The tracer structure, which contains a novel β-fluoroethoxy phosphoester moiety, was designed as an insecticide-chemical nerve agent hybrid to optimize handling and the desired target reactivity. Radiosynthesis of the β-fluoroethoxy tracer is described that utilizes a [¹⁸F]­prosthetic group coupling approach. The imaging utility of the [¹⁸F]­tracer is demonstrated in vivo within rats by the evaluation of its brain penetration and cerebral distribution qualities in the absence and presence of a challenge agent. The tracer effectively penetrates brain and localizes to cerebral regions known to correlate with the expression of the AChE target. Brain pharmacokinetic properties of the tracer are consistent with the formation of an OP-adducted acetylcholinesterase containing the fluoroethoxy tracer group. Based on the initial favorable in vivo qualities found in rat, additional [¹⁸F]­tracer studies are ongoing to exploit the technology to dynamically probe organophosphate mechanisms of action in mammalian live tissues