Reactivation by novel pyridinium oximes of rat serum and skeletal muscle acetylcholinesterase inhibited by organophosphates.

The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.

Pharmacokinetics of three novel pyridinium aldoxime acetylcholinesterase reactivators in female rats.

A platform of novel lipophilic substituted phenoxyalkyl pyridinium oximes was invented to reactivate organophosphate-inhibited acetylcholinesterase. This platform has provided superior efficacy in rats to the current standard of care, 2-PAM, for survival of lethal doses of nerve agent surrogates as well as evidence of brain penetration and neuroprotection. The pharmacokinetics of three of these novel oximes in female rats was studied for comparison to previous data in male rats. Compared to the published half-life of 2-PAM (less than 2 h), the lead novel oxime, Oxime 20, displayed a plasma half-life of about 5 h in both sexes of rats following intramuscular administration. Very few sex differences in pharmacokinetic parameters were apparent. Oxime 20 displayed an increase in brain concentration to plasma concentration over the initial 2 h following intramuscular administration in male rats, with a plateau at 1 h; there were no differences in brain concentrations between the sexes at 2 h. Hepatic metabolism of Oxime 20 was higher in rat microsomes than in human microsomes. The relatively long plasma half-life is likely an important factor in both the enhanced survival and the neuroprotection previously observed for Oxime 20. The metabolism data suggest that the clearance of Oxime 20 could be slower in humans than was observed in rats, which might allow less frequent administration than 2-PAM for therapy of organophosphate acute toxicity. Therefore, the pharmacokinetic data combined with our earlier efficacy data suggest that Oxime 20 has potential as a superior therapeutic for nerve agent poisoning.

Effect of high fat diet on the toxicokinetics and toxicodynamics of chlorpyrifos following acute exposure in male C57BL/6J mice.

Chlorpyrifos (CPS) is one of the most widely used organophosphate (OP) insecticides. The acute neurotoxicity of OPs results from the inhibition of acetylcholinesterase (AChE). However, some OPs also inhibit noncholinergic targets including monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), and carboxylesterase (CES). Data have shown that highly lipophilic OPs, including CPS, have a persistent toxic effect in obese patients. Therefore, the present study was designed to determine the effect of high fat diet (HFD) induced obesity on the disposition of CPS and its detoxified metabolite 3,5,6-trichloro-2-pyridinol (TCP) following acute exposure as well as effects on cholinergic and noncholinergic CPS targets. Male C57BL/6J mice were fed a standard diet (STD) or HFD for 4 weeks, then treated with vehicle or CPS (25 mg/kg) via oral gavage and euthanized postdosing at 0, 3, 6, and 12 h. Following exposure, CPS levels in adipose tissue of HFD fed animals were increased to a greater extent than in STD fed animals, whereas overall hepatic TCP levels were decreased in HFD fed animals. Red blood cell (RBC) AChE and plasma cholinesterase activities were inhibited regardless of diet intake, but inhibition of RBC AChE activity was significantly lower after 3 h in HFD animals. Hepatic CES and FAAH activities were also significantly decreased following CPS exposure regardless of diet. In conclusion, increased time-integrated CPS levels in adipose tissue indicate CPS may possibly form a depot there and may be retained longer in obese animals than in normal animals.

In vitro effect of DDE exposure on the regulation of B-TC-6 pancreatic beta cell insulin secretion: a potential role in beta cell dysfunction and type 2 diabetes mellitus.

Organochlorine compounds (OC) include synthetic insecticides previously used throughout the world before being banned for their adverse effects and environmental persistence; DDT (dichlorodiphenyltrichloroethane) was one of the most widely used. Epidemiological evidence suggests that higher levels of some OC, including metabolites of DDT, such as dichlorodiphenyldichloroethylene (DDE), are associated with type 2 diabetes mellitus (T2D). DDE exposure may affect pancreatic cellular functions associated with glucose control and possibly cause beta cell dysfunction. The in vitro effect of DDE exposure on pancreatic beta cell insulin secretion was investigated using Beta-Tumor Cell-6 (B-TC-6) murine pancreatic beta cells. DDE exposure significantly increased insulin secretion suggesting a role for DDE in altering insulin synthesis and secretion. Reactive oxygen species (ROS) levels were not significantly increased indicating that oxidative stress is not responsible for the DDE-induced insulin secretion. Pancreatic and duodenal homeobox factor-1 (PDX-1) levels were not significantly increased suggesting that DDE exposure does not alter insulin transcription, but prohormone convertase (PC) levels were increased suggesting a role for DDE in altering insulin translation. Based on these in vitro results, DDE may play a role in beta cell dysfunction by affecting mechanisms that regulate insulin secretion but it is not likely to be the major mechanism behind the DDE/T2D epidemiological association.

Effects of novel brain-penetrating oxime acetylcholinesterase reactivators on sarin surrogate-induced changes in rat brain gene expression.

Past assassinations and terrorist attacks demonstrate the need for a more effective antidote against nerve agents and other organophosphates (OP) that cause brain damage through inhibition of acetylcholinesterase (AChE). Our lab has invented a platform of phenoxyalkyl pyridinium oximes (US patent 9,277,937) that demonstrate the ability to cross the blood-brain barrier in in vivo rat tests with a sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP) and provide evidence of brain penetration by reducing cessation time of seizure-like behaviors, accumulation of glial fibrillary acidic protein (GFAP), and hippocampal neuropathology, as opposed to the currently approved oxime, 2-pyridine aldoxime methyl chloride (2-PAM). Using two of the novel oximes (Oximes 1 and 20), this project examined whether gene expression changes might help explain this protection. Expression changes in the piriform cortex were examined using polymerase chain reaction arrays for inflammatory cytokines and receptors. The hippocampus was examined via quantitative polymerase chain reaction for the expression of immediate-early genes involved in brain repair (Bdnf), increasing neurotoxicity (Fos), and apoptosis control (Jdp2, Bcl2l1, Bcl2l11). In the piriform cortex, NIMP significantly stimulated expression for the macrophage inflammatory proteins CCL4, IL-1A, and IL-1B. Oxime 20 by itself elicited the most changes. When it was given therapeutically post-NIMP, the largest change occurred: a 310-fold repression of the inflammatory cytokine, CCL12. In the hippocampus, NIMP increased the expression of the neurotoxicity marker Fos and decreased the expression of neuroprotective Bdnf and antiapoptotic Bcl2l1. Compared with 2-PAM, Oxime 20 stimulated Bcl2l1 expression more and returned expression closer to the vehicle control values.

Inhibition Kinetics of 16 Organophosphorus Pesticides or Their Active Metabolites on Erythrocyte Acetylcholinesterase From Humans and Rats.

Inhibition kinetics assays were conducted with 16 commercial organophosphate (OP) pesticides or their metabolites on acetylcholinesterase (AChE) in erythrocyte "ghost" preparations from 18 individual humans (both sexes; adults, juveniles, and cord blood samples; mixed races/ethnicities) and pooled samples from adult rats (both sexes). A well-established spectrophotometric assay using acetylthiocholine as substrate and a chromogen was employed. The kinetic parameters bimolecular rate constant (ki), dissociation constant (KI), and phosphorylation constant (kp) were calculated for each compound. As expected, a wide range of potencies were displayed among the tested compounds. Statistical analysis of the resultant data indicated no differences in sex, age, or race/ethnicity among the human samples that are unexpected based on chance (4.2% statistically significant out of 48 parameters calculated) and no differences between the sexes in rats. The bimolecular rate constants for 10 of the compounds were not statistically different between rats and humans. The data indicate that, consistent with the high level of conservation of AChE among species and the fact that AChE at different locations within a species arises from the same gene, the inhibition kinetic parameters calculated from rat erythrocyte ghost preparations should be useful in estimating potencies of OP compounds on target AChE in humans. Additionally, the data indicate that differences in sensitivities among individual humans were not apparent.

Reactivation of organophosphate-inhibited serum butyrylcholinesterase by novel substituted phenoxyalkyl pyridinium oximes and traditional oximes.

Organophosphorus compounds (OPs) include nerve agents and insecticides that potently inhibit acetylcholinesterase (AChE), an essential enzyme found throughout the nervous system. High exposure levels to OPs lead to seizures, cardiac arrest, and death if left untreated. Oximes are a critical piece to the therapeutic regimen which remove the OP from the inhibited AChE and restore normal cholinergic function. The current oximes 2-PAM, MMB-4, TMB-4, HI-6, and obidoxime (OBD) have two drawbacks: lack of broad spectrum protection against multiple OP structures and poor brain penetration to protect against OP central neurotoxicity. An alternative strategy to enhance therapy is reactivation of serum butyrylcholinesterase (BChE). BChE is stoichiometrically inhibited by OPs with no apparent toxic result. Inhibition of BChE in the serum followed by reactivation could create a pseudo-catalytic scavenger allowing numerous regenerations of BChE to detoxify circulating OP molecules before they can reach target AChE. BChE in serum from rats, guinea pigs or humans was screened for the reactivation potential of our novel substituted phenoxyalkyl pyridinium oximes, plus 2-PAM, MMB-4, TMB-4, HI-6, and OBD (100µM) in vitro after inhibition by highly relevant surrogates of sarin, VX, and cyclosarin, and also DFP, and the insecticidal active metabolites paraoxon, phorate-oxon, and phorate-oxon sulfoxide. Novel oxime 15 demonstrated significant broad spectrum reactivation of OP-inhibited rat serum BChE while novel oxime 20 demonstrated significant broad spectrum reactivation of OP-inhibited human serum BChE. All tested oximes were poor reactivators of OP-inhibited guinea pig serum BChE. The bis-pyridinium oximes were poor BChE reactivators overall. BChE reactivation may be an additional mechanism to attenuate OP toxicity and contribute to therapeutic efficacy.

In vitro age-related differences in rats to organophosphates.

The mechanism of toxic action for organophosphates (OPs) is the persistent inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and subsequent hyperstimulation of the nervous system. Organophosphates display a wide range of acute toxicities. Differences in the OP's chemistries results in differences in the compound's metabolism and toxicity. Acute toxicities of OPs appear to be principally dependent on compound specific efficiencies of detoxication, and less dependent upon efficiencies of bioactivation and sensitivity of AChE. Serine esterases, such as carboxylesterase (CaE) and butyrylcholinesterase (BChE), play a prominent role in OP detoxication. Organophosphates can stoichiometrically inhibit these enzymes, removing OPs from circulation thus providing protection for the target enzyme, AChE. This in vitro study investigated age-related sensitivity of AChE, BChE and CaE to twelve structurally different OPs in rat tissues. Sensitivity of esterases to these OPs was assessed by inhibitory concentration 50s (IC50s). The OPs displayed a wide range of inhibitory potency toward AChE with IC50s in the low nM-µM range with no differences among ages; however, the CaE IC50s generally increased with age reflecting greater protection in adults. These results suggest age-related differences in acute toxicities of OPs in mammals are primarily a result of their detoxication capacities.

Novel pyridinium oximes enhance 24-h survivability against a lethal dose of nerve agent surrogate in adult female rats.

Our laboratory has developed novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) designed to more efficiently penetrate the central nervous system to enhance survivability and attenuate seizure-like signs and neuropathology. Previous studies with male Sprague-Dawley rats indicated that survivability was enhanced against the nerve agent (sarin) surrogate, 4-nitrophenyl isopropyl methylphosphonate (NIMP). In this study, female adult Sprague-Dawley rats, tested specifically in diestrus, were challenged subcutaneously with lethal concentrations of NIMP (0.6 mg/kg). After development of seizure-like behavior and other signs of cholinergic toxicity, human equivalent dosages of atropine (0.65 mg/kg) and one of four oximes (2-PAM, or novel oxime 15, 20, or 55; 0.146 mmol/kg) or Multisol vehicle was administered alone or in binary oxime combinations intramuscularly. Animals were closely monitored for signs of cholinergic toxicity and 24 h survivability. Percentages of animals surviving the 24 h NIMP challenge dose were 35 % for 2-PAM and 55 %, 70 %, and 25 % for novel oximes 15, 20, and 55, respectively. Improvements in survival were also observed over 2-PAM alone with binary combinations of 2-PAM and either oxime 15 or oxime 20. Additionally, administration of novel oximes decreased the duration of seizure-like behavior as compared to 2-PAM suggesting that these oximes better penetrate the blood-brain barrier to mitigate central nervous system hypercholinergic activity. Efficacies were similar between females and previously reported males. These data indicate that the novel pyridinium oximes enhance survivability against lethal OP toxicity as compared to 2-PAM in adult female rats.

Oxime-mediated reactivation of organophosphate-inhibited acetylcholinesterase with emphasis on centrally-active oximes.

This review provides an overview of the global research leading to the large number of compounds developed as reactivators of acetylcholinesterase inhibited by a variety of organophosphate compounds, most of which are nerve agents but also some insecticides. A number of these organophosphates are highly toxic and effective therapy by reactivators contributes to saving lives. Two major challenges for more effective therapy with reactivators are identification of a broad spectrum reactivator efficacious against a variety of organophosphate structures, and a reactivator that can cross the blood-brain barrier to protect the brain. The most effective of the reactivators developed are the nucleophilic pyridinium oximes, which bear a permanent positive charge from the quaternary nitrogen in the pyridinium ring. The permanent positive charge retards the oximes from crossing the blood-brain barrier and therefore restoration of normal cholinergic function in the brain is unlikely. A number of laboratories have developed nucleophiles, mostly oximes, that are theorized to cross the blood-brain barrier by several strategies. At the present time, no reactivator is optimally broad spectrum across the wide group of organophosphate chemistries. Some oximes, including the substituted phenoxyalkyl pyridinium oximes invented by our laboratories, have the potential to provide neuroprotection in the brain and show evidence of efficacy against both nerve agent and insecticidal chemistries, so these novel oximes have promise for future development. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Central neuroprotection demonstrated by novel oxime countermeasures to nerve agent surrogates.

Oximes remain a long-standing element of the therapy for nerve agents, organophosphates (OPs) that poison by inhibiting the enzyme acetylcholinesterase (AChE), resulting in hypercholinergic activity both centrally and peripherally. Oximes, such as the pyridinium oxime pralidoxime (2-PAM) in the United States, can reactivate the inhibited AChE and restore cholinergic function. However, there are several drawbacks to the current oximes; one of them, the inability of these oximes to effectively enter the brain, is the subject of study by several laboratories, including ours. Our laboratory invented a platform of substituted phenoxyalkyl pyridinium oximes that were tested against highly relevant surrogates of the nerve agents, sarin and VX. Using high sublethal dosages of the OPs, the novel oximes were observed to attenuate seizure-like behavior in rats and to reduce the levels of glial fibrillary acidic protein (an indicator of glial scarring) to control levels, in contrast to levels observed with 2-PAM or no oxime therapy. Using lethal levels of surrogates, some novel oximes protected against lethality compared with 2-PAM, shortened the time to cessation of seizure-like behavior (from 8+ to 6 h), and protected the brain neurons. Therefore, some of these novel oximes are showing exceptional promise alone or in combination with 2-PAM as therapeutics against nerve agent toxicity.

Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX.

A novel oxime platform, the substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937), was invented at Mississippi State University with an objective of discovering a brain-penetrating antidote to highly potent organophosphate anticholinesterases, such as the nerve agents. The goal was reactivation of inhibited brain acetylcholinesterase to attenuate the organophosphate-induced hypercholinergic activity that results in glutamate-mediated excitotoxicity and neuropathology. The currently approved oxime antidote in the US, 2-PAM, cannot do this. Using highly relevant surrogates of sarin and VX that leave acetylcholinesterase phosphylated with the same chemical moiety as their respective nerve agents, in vitro screens and in vivo tests in rats were conducted to identify the most efficacious members of this platform. The most promising novel oximes provided 24-h survival of lethal level surrogate exposure better than 2-PAM in almost all cases, and two of the oximes shortened the time to cessation of seizure-like behavior while 2-PAM did not. The most promising novel oximes attenuated neuropathology as indicated by immunohistochemical stains for both glia and neurons, while 2-PAM did not protect either glia or neurons. These results strongly suggest that these novel oximes can function within the brain to protect it, and therefore show great promise as potential future nerve agent antidotes.

Association of serum levels of p,p'- Dichlorodiphenyldichloroethylene (DDE) with type 2 diabetes in African American and Caucasian adult men from agricultural (Delta) and non-agricultural (non-Delta) regions of Mississippi.

Epidemiological associations were reported in several studies between persistent organochlorine organic pollutants and type 2 diabetes mellitus (T2D). Mississippi is a highly agricultural state in the USA, particularly the Delta region, with previous high usage of organochlorine (OC) insecticides such as p,p'- dichlorodiphenyltrichloroethane (DDT). In addition, there is a high proportion of African Americans who display elevated prevalence of T2D. Therefore, this State provides an important dataset for further investigating any relationship between OC compounds and metabolic diseases. The aim of this study was to assess whether soil and serum levels of OC compounds, such as p,p'- dichlorodiphenyldichloroethylene (DDE), arising from the heavy historical use of legacy OC insecticides, might serve as an environmental public health indicator for T2D occurrence. Soil samples from 60 Delta and 60 non-Delta sites randomly selected were analyzed for the presence of OC compounds. A retrospective cohort study of adult men (150 from each region) was recruited to provide a blood sample for OC compound quantitation and select demographic and clinical information including T2D. Using multivariable logistic regression, an association was found between increasing serum DDE levels and T2D occurrence in non-Delta participants (those subjects with lower serum DDE levels), as opposed to Delta participants (individuals with higher serum DDE levels). Thus, while there was a relationship between serum DDE levels and T2D in those with lower burdens of DDE, the lack of association in those with higher levels of DDE indicates a complex non-monotonic correlation between serum DDE levels and T2D occurrence complicating the goal of finding a public health marker for T2D. Abbreviations: BMI, body mass index; CVD, cardiovascular disease; CDC, Center for Disease Control, United States of America; DDE, p,p'- dichlorodiphenyldichloroethylene; DDT, p,p'- dichlorodiphenyltrichloroethane; GC/MS, gas chromatography/mass spectrometry; GIS, geographic information system; GPS, global positioning system; HDL, high-density lipoprotein; HTN, hypertension; IDW, inverse distance weighting; IRB, Institutional Review Board; LDL, low-density lipoprotein; LOQ, limit of quantitation; NHANES, National Health and Nutrition Examination Surveys; POPs, persistent organic pollutants; OC, organochlorine; PCB, polychlorinated biphenyl; SIM, single-ion monitoring; T2D, type 2 diabetes mellitus; USA, United States of America.

Novel Brain-Penetrating Oxime Acetylcholinesterase Reactivators Attenuate Organophosphate-Induced Neuropathology in the Rat Hippocampus.

Organophosphate (OP) anticholinesterases cause excess acetylcholine leading to seizures which, if prolonged, result in neuronal damage in the rodent brain. Novel substituted phenoxyalkyl pyridinium oximes have previously shown evidence of penetrating the rat blood-brain barrier (BBB) in in vivo tests with a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the active metabolite of the insecticide parathion, paraoxon (PXN), by reducing the time to cessation of seizure-like behaviors and accumulation of glial fibrillary acidic protein, whereas 2-PAM did not. The neuroprotective ability of our lead oximes (15, 20, and 55) was tested using NeuN, Nissl, and Fluoro-Jade B staining in the rat hippocampus. Following lethal-level subcutaneous challenge with NIMP or PXN, rats were intramuscularly administered a novel oxime or 2-PAM plus atropine and euthanized at 4 days. There were statistically significant increases in the median damage scores of the NeuN-stained NIMP, NIMP/2-PAM, and NIMP/Oxime 15 groups compared with the control whereas the scores of the NIMP/Oxime 20 and NIMP/Oxime 55 were not significantly different from the control. The same pattern of statistical significance was observed with PXN. Nissl staining provided a similar pattern, but without statistical differences. Fluoro-Jade B indicated neuroprotection from PXN with novel oximes but not with 2-PAM. The longer blood residence times of Oximes 20 and 55 compared with Oxime 15 might have contributed to their greater efficacy. These results suggest that novel oximes 20 and 55 were able to penetrate the BBB and attenuate neuronal damage after NIMP and PXN exposure, indicating potential broad-spectrum usefulness.

Neuroprotection From Organophosphate-Induced Damage by Novel Phenoxyalkyl Pyridinium Oximes in Rat Brain.

The nerve agents are extremely toxic organophosphates which lead to massive inhibition of acetylcholinesterase (AChE) in both the central and peripheral nervous systems. The currently approved pyridinium oxime reactivators of organophosphate-inhibited AChE (eg, 2-PAM in the United States) cannot penetrate the blood-brain barrier because of the permanent positive charge in the pyridinium ring. Therefore these current oximes cannot rescue inhibited AChE in the brain. Our laboratories have invented and patented a platform of substituted phenoxyalkyl pyridinium oximes that have been tested for efficacy as therapy within the brains of adult male rats which were challenged with a high sublethal dosage of highly relevant surrogates of sarin (nitrophenyl isopropyl methylphosphonate, NIMP) and VX (nitrophenyl ethyl methylphosphonate, NEMP). The histochemical astrocyte marker glial fibrillary acidic protein (GFAP) was investigated as an indication of neuropathology in two brain regions, the piriform cortex and the dentate gyrus of the hippocampus, which are regions known to be damaged by nerve agent toxicity. Rats treated with either NIMP or NEMP without therapy or with NIMP or NEMP plus 2-PAM therapy showed similar increases in GFAP compared with vehicle controls. However, the rats challenged with NIMP or NEMP plus therapy with our novel Oxime 20 (either a bromide or a mesylate salt) showed GFAP levels statistically undistinguishable from controls. These data provide highly supportive functional evidence of novel oxime entry into the brain. These novel oximes have the potential to provide central neuroprotection from organophosphate anticholinesterase-induced damage, which is a characteristic not displayed by most pyridinium oximes.

A case-control study: The association of serum paraoxonase 1 activity and concentration with the development of type 2 diabetes mellitus.

BACKGROUND: A longitudinal study assessed serum paraoxonase 1 (PON1) activity and concentration as affected by age and as associated with the development of type 2 diabetes (T2D). PON1's recently established physiological function is the hydrolysis of lipolactones in oxidized LDL particles. METHODS: Serum samples and clinical data collected and stored at different time points over a 20-year interval in the Air Force Health Study were analysed. PON1 activity and concentration and C-reactive protein concentration in samples from the same individuals 20 years apart were compared using a paired t test (n = 159). A case-control study design and multivariable logistic regression analysis assessed the association of PON1's activity and concentration with the subsequent development of T2D (n = 222 and α = 0.10). RESULTS: No difference with age was found in PON1 activity assessed using 3 substrates, paraoxon (P = 0.897), phenyl acetate (P = 0.994), and dihydrocoumarin (P = 0.505), or PON1 serum concentration (P = 0.357). C-reactive protein concentration increased 0.7 mg/L (P = 0.004) over the 20-year interval. Lower PON1 activity assayed with phenyl acetate (P = 0.015, OR = 1.25 per 1000 U/L decrease) was associated with an increased risk of developing T2D as was a lower PON1 serum concentration (P = 0.004, OR = 1.72 per 2 µmol/L decrease). PON1 activity assayed with paraoxon (P = 0.681) or dihydrocoumarin (P = 0.136) was not associated with the development of T2D. CONCLUSIONS: Lower PON1 activity and concentration were associated with an increased risk of developing T2D when adjusted for many of the common risk markers for T2D previously identified. Thus, PON1 may have merit as a biomarker for the development of T2D.

Effects of Chlorpyrifos or Methyl Parathion on Regional Cholinesterase Activity and Muscarinic Receptor Subtype Binding in Juvenile Rat Brain.

The effects of developmental exposure to two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in preweanling rat brain. Animals were orally gavaged daily with low, medium, and high dosages of the insecticides using an incremental dosing regimen from postnatal day 1 (PND1) to PND20. On PND12, PND17 and PND20, the cerebral cortex, corpus striatum, hippocampus, and medulla-pons were collected for determination of ChE activity, total mAChR density, and the density of the individual mAChR subtypes. ChE activity was inhibited by the medium and high dosages of CPF and MPS at equal levels in all four brain regions at all three ages examined. Exposure to both compounds decreased the levels of the M1, M2/M4, and M3 subtypes and the total mAChR level in all brain regions, but the effects varied by dosage group and brain region. On PND12, only the high dosages induced receptor changes while on PND17 and PND20, greater effects became evident. In general, the effects on the M1 subtype and total receptor levels appeared to be greater in the cerebral cortex and hippocampus than in the corpus striatum and medulla-pons. This did not appear to be the case for the M2/M4 and M3 subtypes effects. The differences between CPF and MPS were minimal even though in some cases, CPF exerted statistically greater effects than MPS did. In general, repeated exposure to organophosphorus insecticides can alter the levels of the various mAChR subtypes in various brain regions which could induce perturbation in cholinergic neurochemistry during the maturation of the brain regions.

In Vitro effect of DDE exposure on the regulation of lipid metabolism and secretion in McA-RH7777 hepatocytes: A potential role in dyslipidemia which may increase the risk of type 2 diabetes mellitus.

Organochlorine compounds (OC), such as the legacy insecticides, were widespread environmental contaminants. OC including dichlorodiphenyldichloroethylene (DDE), a metabolite of the insecticide DDT, have an epidemiological association with type 2 diabetes mellitus (T2D) and may play a role in risk factors that contribute to T2D such as dyslipidemia. The liver, a potential target for DDE, plays a role in dyslipidemia. The in vitro effect of DDE on hepatocyte lipid metabolism and secretion was investigated using McArdle-RH7777 (McA) rodent hepatoma liver cells. When stimulated by the free fatty acid oleic acid (OA), DDE increased the secretion of apolipoprotein B (ApoB) suggesting a role for DDE in increasing lipid secretion. Intracellular protein levels of microsomal triglyceride transfer protein (MTP) were increased while sortilin-1 (Sort-1) levels were decreased suggesting a role for DDE in increasing lipid transport and decreasing lipid degradation. Neutral lipids such as intracellular triglycerides (TG) were decreased suggesting that DDE may alter lipid accumulation in liver cells. DDE may play a role in dyslipidemia by affecting mechanisms that regulate lipid metabolism and secretion. These in vitro results on biochemical markers of liver cell dyslipidemia support the concept that DDE exposure may play a role in the dyslipidemia frequently observed in T2D.

Efficacy of novel phenoxyalkyl pyridinium oximes as brain-penetrating reactivators of cholinesterase inhibited by surrogates of sarin and VX.

Pyridinium oximes are strong nucleophiles and many are effective reactivators of organophosphate-inhibited cholinesterase (ChE). However, the current oxime reactivators are ineffective at crossing the blood-brain barrier and reactivating brain ChE in the intact organism. Our laboratories have developed a series of substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937 B2) with the goal of identifying reactivators effective in crossing the blood-brain barrier. The first 35 of the series were found to have similar in vitro efficacy as reactivators of ChE inhibited by a sarin surrogate (phthalimidyl isopropyl methylphosphonate, PIMP) or a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP) in bovine brain preparations as previously observed in rat brain preparations. A number of these novel oximes have shown the ability to decrease the level of ChE inhibition in the brains of rats treated with a high sublethal dosage of either a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the VX surrogate NEMP. Levels of reactivation at 2 h after oxime administration were up to 35% while the currently approved therapeutic, 2-PAM, yielded no reduction in brain ChE inhibition. In addition, there was evidence of attenuation of seizure-like behavior with several of the more effective novel oximes, but not 2-PAM. Therefore these novel oximes have demonstrated an ability to reactivate inhibited ChE in brain preparations from two species and in vivo data support their ability to enter the brain and provide a therapeutic action. These novel oximes have the potential to be developed into improved antidotes for nerve agent therapy.

In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).

BACKGROUND: Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. METHODS: To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences enzyme reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion. RESULTS: The permeability of 2-PAM is about 2 × 10(-6) cm s(-1) in MDCK cells and about 1 × 10(-6) cm s(-1) in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps. CONCLUSIONS: The low permeability explains poor brain penetration of 2-PAM and therefore the slow enzyme reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.