Biomedical subjects

M Ehrich

Publications and source records attributed to M Ehrich.

At least 37 records · Page 2Linked to original sources

PubMed1998

[Benzodiazepine consumption in Hvalsø. Can it be further reduced in a region in which earlier intervention reduced consumption by 38 per cent?].

Hvalsø is a country town with six general practitioners in five practices. In 1988, as a result of a campaign influencing both doctors and patients, a 38% reduction in the consumption of benzodiazepines, measured as the number of prescribed doses, was achieved. This reduction still persists. We have now attempted to reduce consumption even further by directly influencing the individual users. We gave them written information, insisted on personal attendance for each prescription renewal, and, for use at these consultations, introduced a new benzodiazepine journal for 60% of the users. Registration of the prescribed amounts of benzodiazepines was performed over two three-month periods, before and after the intervention. The final registration was made six months after the intervention period. The number of prescriptions, number of prescriptions per 1000 patients and the number of users remained unchanged. A 20% reduction in the amount of prescribed sedatives (hypnotics) and a 7% reduction in prescribed minor tranquillizers was achieved because of fewer doses per prescription. We conclude, that we did not manage to change the patients' behaviour, expressed as the number of prescriptions per 1000 patients, but the doctors were influenced to write out fewer doses per prescription. Important reductions in consumption may be achieved in primary interventions.

Attitude to Health↗

PubMed1998

Inhibition of carboxylesterases in SH-SY5Y human and NB41A3 mouse neuroblastoma cells by organophosphorus esters.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

Acetylcholinesterase↗

PubMed1998

Common mechanism of toxicity: a case study of organophosphorus pesticides.

The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.

Animals↗

PubMed1998

Alterations of cytoskeletal tau protein of SH-SY5Y human neuroblastoma cells after exposure to MPTP.

In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 10(-3) to 10(-4) M for 2 to 5 days) increased the expression of microtubule-associated tau protein in both the supernatant and pellet fractions of lysed SH-SY5Y human neuroblastoma cells. The western blot using anti-tau-1 antibodies demonstrated that the cells contained at least six isoforms of tau proteins, five with molecular weights from 45 to 62 kD. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers coding whole length tau protein further confirmed the presence of tau in SH-SY5Y cells. The PCR product of tau in SH-SY5Y cells had approximately 1050 base pairs. MPTP caused an increased expression of the PCR product of tau, suggesting that the toxicant caused an increase in mRNA coding the tau protein. The expression of cytoskeletal tau protein may, therefore, provide a marker for MPTP neurotoxicity in SH-SY5Y cells.

Cytoskeletal Proteins↗

PubMed1997

Acetylcholinesterase and neuropathy target esterase inhibitions in neuroblastoma cells to distinguish organophosphorus compounds causing acute and delayed neurotoxicity.

The differential inhibition of the target esterases acetylcholinesterase (AChE) and neuropathy target esterase (NTE, neurotoxic esterase) by organophosphorus compounds (OPs) is followed by distinct neurological consequences in exposed subjects. The present study demonstrates that neuroblastoma cell lines (human SH-SY5Y and murine NB41A3) can be used to differentiate between neuropathic OPs (i.e., those inhibiting NTE and causing organophosphorus-induced delayed neuropathy) and acutely neurotoxic OPs (i.e., those highly capable of inhibiting AChE). In these experiments, concentration-response data indicated that the capability to inhibit AChE was over 100x greater than the capability to inhibit NTE for acutely toxic, nonneuropathic OPs (e.g., paraoxon and malaoxon) in both cell lines. Inhibition of AChE was greater than inhibition of NTE, without overlap of the concentration-response curves, for OPs which are more likely to cause acute, rather than delayed, neurotoxic effects in vivo (e.g., chlorpyrifos-oxon, dichlorvos, and trichlorfon). In contrast, concentrations inhibiting AChE and NTE overlapped for neuropathy-causing OPs. For example, apparent IC50 values for NTE inhibition were less than 9.6-fold the apparent IC50 values for AChE inhibition when cells were exposed to the neuropathy-inducing OPs diisopropyl phosphorofluoridate, cyclic tolyl saligenin phosphate, phenyl saligenin phosphate, mipafox, dibutyl dichlorovinyl phosphate, and di-octyl-dichlorovinyl phosphate. In all cases, esterase inhibition occurred at lower concentrations than those needed for cytoxicity. These results suggest that either mouse or human neuroblastoma cell lines can be considered useful in vitro models to distinguish esterase-inhibiting OP neurotoxicants.

Animals↗

PubMed1997

Synaptophysin immunoreactive axonal swelling in p-bromophenylacetylurea-induced neuropathy.

A single intraperitoneal dose of 300 mg/kg of p-bromophenylacetylurea (BPAU) induced progressive distal neuropathy in rats, prominently involving peripheral nerves and long central nervous system myelinated tracts such as the fasciculus gracilis and spinocerebellar pathways. Clinical signs assessed using a Functional Observational Battery (FOB) and in-cage observation included weakness and deficits in motor and sensory integration, definitively noted on post-dosing day 7. The signs were more pronounced upon repetition of the FOB on post-dosing day 12. The neuropathological substrate of these signs was a progressive axonopathy with regional swelling, leading to Wallerian-like degeneration of affected myelinated fibers. Immunocytochemical staining for synaptophysin revealed often striking increase in immunoreactivity for this synaptic vesicle glycoprotein in swollen and otherwise injured axons. Such accumulations were considered consistent with interruption of anterograde (and possibly retrograde) fast axonal transport systems secondary to toxicant-induced nerve fiber breakdown.

Animals↗

PubMed1997

Cell culture models of interspecies selectivity to organophosphorous insecticides.

In toxicology, the need to reduce uncertainties in human risk assessment is met by understanding why species and individuals within that species respond differently to chemical exposure. This kind of information is needed when extrapolating data from experimental (i.e., whole animal) systems to the human condition in terms of risk assessment. In 1993 the Neurotoxicology Division of the Environmental Protection Agency funded several investigators to examine this phenomenon (i.e., interspecies selectivity) using cell culture models. Organophosphorous (OP) insecticides were examined since they are characterized by an extremely divergent interspecies response. In 1995, a symposium entitled Novel Insights into Chemical Neurotoxicity, sponsored by the Society for In Vitro Biology featured this research. In it, a historical overview of the phenomenon of interspecies selectivity to OP insecticides was given, current explanations for it were discussed and contemporary in vitro models being used to explain it, were described. Data from these studies have helped to redefine the underlying mechanisms that characterize and influence the cross-species response to insecticides. These experiments have refocused the explanation of this phenomenon to include cellular metabolism, target enzyme baseline activities, and receptor-mediated electrophysiological and second-messenger events. Several investigators on this panel also reported on the use of subcellular markers (e.g., target esterases, second messengers, ionic fluxes) to differentiate neuropathy-causing OP compounds from acetylcholinesterase inhibitors. After these presentations, technical considerations used in the designed of in vitro neurotoxicity studies were discussed.

Animals↗

PubMed1997

Cytotoxic effects of MPTP on SH-SY5Y human neuroblastoma cells.

Morphological and metabolic endpoints were used to evaluate MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity to SH-SY5Y human neuroblastoma cells. After 8 hours of exposure, MPTP was found to affect cell viability only at a very high concentration (3 x 10(-3) M), but its metabolite MPP+ could decrease viability at 10(-4) M. MPTP, via its metabolite MPP+, inhibited NADH dehydrogenase activity when concentrations exceeded 10(-4) M (for MPP+ 10(-5)M). The Ki were 2.4 x 10(-3) M and 3 x 10(-4)M for MPTP and MPP+, respectively. MPTP at concentrations greater than 10(-4) M altered cell morphology as early as one hour after exposure. These changes included formation of cell surface blebs and attenuated neurites. After 8 hours at 10(-3) M and 24 hrs at 10(-4) M, MPTP caused ultrastructural changes of mitochondria with increased electron-density of the matrix and disorganization of cristae, as well as abnormal aggregation of filamentous material of the cytoskeleton. Because these changes of structure and function took place at concentrations lower than those needed to affect cell viability, they may play a role in MPTP neurotoxicity in SH-SY5Y cell culture.

1-Methyl-4-phenylpyridinium↗

PubMed1996

Subchronic delayed neurotoxicity evaluation of jet engine lubricants containing phosphorus additives.

Synthetic polyol-based lubricating oils containing 3% of either commercial tricresyl phosphate (TCP), triphenylphosphorothionate (TPPT), or butylated triphenyl phosphate (BTP) additive were evaluated for neurotoxicity in the adult hen using clinical, biochemical, and neuropathological endpoints. Groups of 17-20 hens were administered the oils by oral gavage at a "limit dose" of 1 g/kg, 5 days a week for 13 weeks. A group of positive control hens was included which received 7.5 mg/kg of one isomer of TCP (tri-ortho-cresyl phosphate, TOCP) on the same regimen, with an additional oral dose of 500 mg/kg given 12 days before the end of the experiment. A negative control group received saline. Neurotoxic esterase (NTE) activity in brain and spinal cord of hens dosed with the lubricating oils was not significantly different from saline controls after 6 weeks of treatment. After 13 weeks of dosing, NTE was inhibited 23 to 34% in brains of lubricant-treated hens. Clinical assessments of walking ability did not indicate any differences between the negative control group and lubricant-treated hens. Moreover, neuropathological examination revealed no alterations indicative of organophosphorus-induced delayed neuropathy (OPIDN). In hens treated with the positive control, significant inhibition of NTE was observed in brain and spinal cord at both 6 and 13 weeks of dosing; this group also demonstrated clinical impairment and pathological lesions indicative of OPIDN. In conclusion, the results of the present study indicated that synthetic polyol-based lubricating oils containing up to 3% TCP, TPPT, or BTP had low neurotoxic potential and should not pose a hazard under realistic conditions of exposure.

Acetylcholinesterase↗

PubMed1996

Biotransformation of the MPTP analog trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetra- hydropyridine to a fluorescent pyridinium metabolite by intact neuroblastoma cells.

The possibility of imaging monoamine oxidase (MAO) containing neurons through the MAO-mediated conversion of the nonfluorescent tetrahydropyridine compound trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetrahydro pyridine (t-THP) to the corresponding fluorescent trans-1-methyl-4-[4-dimethylaminophenylethenyl]pyridinium species (t-P+) was examined with the aid of human neuroblastoma cells (SH-SY5Y). Fluorescence microscopy and fluorescence measurements established the intracellular formation of a fluorescent species with maximal excitation/emission wavelengths of 485/620 and 530/620 nm corresponding to the fluorescence characteristics of synthetic t-P+. An independent assay confirmed the presence of both MAO-A and MAO-B in these cells. As expected, the development of the fluorescence was inhibited by both clorgyline (an MAO-A inhibitor) and deprenyl (an MAO-B inhibitor). Cytotoxic effects, as determined by trypan blue dye exclusion for viability and by the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay for mitochondrial integrity, were not observed in cells incubated with concentrations of t-THP as high as 10(-3) M for 4 hr. The results from these studies with a neuronal cell line of human origin suggest: (1) that SH-SY5Y cells metabolize and, therefore, can be used for study of tetrahydropyridine compounds in vitro, and (2) that t-THP may be a useful agent to monitor neurodegenerative processes in MAO-rich neurons, including the dopaminergic nigrostriatal neurons that are damaged by the parkinsonian-inducing tetrahydropyrridine MPTP. The potential advantage of using t-THP over related imaging techniques is the possibility of assessing neuronal function by an in vivo processing of the reporter molecule rather than by postmortem immunofluorescent or formaldehyde-based procedures.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1996

Catecholamine concentrations and contractile responses of isolated vessels from hens treated with cyclic phenyl saligenin phosphate or paraoxon in the presence or absence of verapamil.

Blood samples and vascular segments from the ischiadic artery of hens treated with either cyclic phenyl saligenin phosphate (PSP; 2.5 micrograms/kg, im) or paraoxon (PXN; 0.1 micrograms/kg, im) in the presence or absence of verapamil, a calcium channel antagonist (7 micrograms/kg, im, given 4 consecutive days beginning the day before PSP or PXN administration), were examined 1, 3, 7, and 21 d after PSP or PXN administration in order to determine the contribution of catecholamines and peripheral blood vessel physiology and morphology to organophosphorus-induced delayed neuropathy (OPIDN). The levels of plasma catecholamines were measured by high-performance liquid chromatograpy (HPLC) and indicated a different effect with PSP, which causes OPIDN, and PXN, which does not. PSP treatment elevated the levels of norepinephrine and epinephrine throughout the study, while PXN treatment depressed the levels of these catecholamines. Verapamil treatment attenuated the OP response by approximately 50% for both compounds. Ischiadic vessel segments were isolated from OP-treated hens and perfused at a constant flow rate of 12 ml/min, then examined for their response to potassium chloride (KCl, 3 x 10(-3) M), acetylcholine (ACh), phenylephrine (PE), an alpha 1 adrenergic agonist, and salbutamol (SAL), a beta 2 adrenergic agonist. Agents were delivered in concentrations of 10(-8) to 10(-3) M. Vascular segments did not respond to ACh or SAL at any concentration used. Vessels displayed a significant reduction in contractile response to both KCl (3 x 10(-3) M) and PE (10(-8) to 10(-3) M) 3 and 21 d after exposure to either PSP or PXN. This reduced response was not altered by the presence of verapamil. Innervation of the peripheral vasculature was unchanged after OP treatment. This study indicates that plasma catecholamine levels could be differentially altered by treatment with OPs that do and do not cause OPIDN and suggests that the alterations involve intracellular calcium. In contrast, vascular response of the ischiadic artery was altered following OP treatment, but the effect was not specific for the neuropathy-inducing OP, PSP, and response was not mediated by Ca 2+, nor was it the result of autonomic nerve deterioration.

Acetylcholine↗

PubMed1995

Comparison of the relative inhibition of acetylcholinesterase and neuropathy target esterase in rats and hens given cholinesterase inhibitors.

Inhibition of neuropathy target esterase (NTE, neurotoxic esterase) and acetylcholinesterase (AChE) activities was compared in brain and spinal cords of adult While Leghorn hens and adult male Long Evan rats 4-48 hr after administration of triortho-tolyl phosphate (TOTP po, 50-500 mg/kg to hens; 300-1000 mg/kg to rats), phenyl saligenin phosphate (PSP im 0.1-2.5 mg/kg to hens; 5-24 mg/kg to rats), mipafox (3-30 mg/kg ip to hens and rats), diisopropyl phosphorofluoridate (DFP sc, 0.25-1.0 mg/kg to hens; 1-3 mg/kg to rats), dichlorvos (5-60 mg/kg ip to hens; 600-2000 mg/kg to rats), and carbaryl (300-560 mg/kg ip to hens; 30-170 mg/kg to rats). Inhibitions of NTE and AChE were dose-related after administration of all compounds to both species. Hens and rats given TOTP, PSP, mipafox, and DFP demonstrated delayed neuropathy 3 weeks later, with spinal cord lesions and clinical signs more notable in hens. Ratios of NTE/AChE inhibition in hen spinal cord, averaged over the doses used, were 2.6 after TOTP, 5.2 after PSP, 1.3 after mipafox, and 0.9 after DFP, which contrast with 0.53 after dichlorvos, 1.0 after malathion, and 0.46 after carbaryl. Rat NTE/AChE inhibition ratios were 0.9 after TOTP, 2.6 after PSP, 1.0 after mipafox, 0.62 after DFP, 1.3 after dichlorvos, 2.2 after malathion, and 1.1 after carbaryl. The lower NTE/AChE ratios in rats given dosages of the four organophosphorus compounds that caused delayed neuropathy interferred with survival, an effect that was not a problem in hens.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1995

Possible involvement of a neurotrophic factor during the early stages of organophosphate-induced delayed neurotoxicity.

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neurotoxic esterase (NTE). We hypothesized that a trophic factor may be produced in situ shortly after exposure to the OP which participates in the progression of OPIDN. To bioassay for such a growth-modulating factor(s), we treated chickens with the neuropathic agents diisopropylfluorophosphate (DFP) or cyclic phenyl saligenin phosphate (PSP), with or without phenylmethylsulfonyl fluoride (PMSF, a chemical which markedly modifies OPIDN). Soluble extracts of cervical spinal cord (a region of the nervous system which degenerates with OPIDN) were collected 24 h later and these were incubated with human neuroblastoma SY5Y cells in culture. The cells were allowed to grow for another 6 days and observed for changes in morphology and growth. After 3 days in culture, tissue extracts from OP-treated chickens caused SY5Y cells to begin to elongate and extend processes (neurites), similar to cells treated with nerve growth factor (1 microgram/ml). Extracts from chickens not receiving OP had no or minimal effects on cell morphology. In addition, extracts from chickens in which OPIDN was prevented by pretreatment with PMSF did not cause the marked extension of cell processes exhibited after exposure of SY5Y cells to extracts from chickens given regimens known to cause OPIDN. In parallel-treated animals. DFP and PSP caused clinical dysfunction characteristic of OPIDN, PMSF posttreatment markedly amplified the clinical deficits and PMSF pretreatment prevented OPIDN. In vivo DFP treatment also caused a marked reduction in the activity of the growth-related enzyme ornithine decarboxylase (ODC) in spinal cord but DFP was without effect on ODC activity in vitro (up to 1 mM final concentration). Characterization of this growth-modulating factor(s) may aid in the elucidation of pathological mechanisms of OPIDN.

Animals↗

PubMed1995

Effect of cyclic phenyl saligenin phosphate and paraoxon treatment on vascular response to adrenergic and cholinergic agents in hens.

The response of peripheral blood vessels to adrenergic and cholinergic agonists was examined 1, 3, 7, and 21 d after hens were treated with a single intramuscular injection of 2.5 mg/kg cyclic phenyl saligenin phosphate (PSP) or 0.10 mg/kg paraoxon (PXN). These two organophosphates (OPs) cause different clinical effects in exposed animals, as PSP causes organophosphate-induced delayed neuropathy (OPIDN) and PXN causes acute poisoning through inhibition of acetylcholinesterase. For these studies, the ischiadic artery was cannulated both prograde and retrograde and the blood was shunted through a pump to maintain a constant flow. Alterations in pressure measured at the pump outflow were used to indicate changes in limb vascular resistance. Dose-response curves were generated for the response to intravenous administration of acetylcholine (ACh), phenylephrine (PE), or salbutamol (SAL) (10(-8) to 10(-4) mol/kg). Acetylcholine at 10(-8) to 10(-7) mol/kg caused an increase in vascular resistance, whereas concentrations of 10(-5) to 10(-4) mol/kg caused a decrease in vascular resistance in hens given PSP 1 and 3 d previously. The response of PXN-treated hens to ACh was not significantly altered from that of vehicle-treated hens. The resistance generated in response to PE, an alpha 1-adrenergic agonist, in PSP-treated hens was greater than levels in vehicle-treated hens on d 1 and 3 and greater than the response seen in hens treated with PXN. Salbutamol, a beta 2-adrenergic agonist, at concentrations of 10(-7) to 10(-4) mol/kg caused an increase in resistance 1 and 3 d after PSP and a decrease on d 7. The responses to SAL were different in PXN-treated hens, as these hens demonstrated a lesser increase in resistance at concentrations of 10(-8) to 10(-7) mol/kg and a decrease in resistance at 10(-5) to 10(-4) mol/kg 1 d after administration of PXN. These observations indicate that response to vasoactive agents is altered in OP-treated hens and that responses differ between a compound capable of causing OPIDN (PSP) and a compound that only causes acute effects (PXN).

Adrenergic Agents↗

PubMed1995

Using neuroblastoma cell lines to address differential specificity to organophosphates.

1. Organophosphates can cause acute toxicity, which follows inhibition of acetylcholinesterase (AChE), or delayed neuropathy, which follows inhibition of neuropathy target esterase (NTE). 2. Human neuroblastoma SH-SY5Y cells contain AChE and NTE. 3. Organophosphates actively able to inhibit AChE in animal models inhibited AChE in neuroblastoma cells. 4. Inhibition of NTE in neuroblastoma cells could identify active organophosphates capable of causing delayed neuropathy in animal models and distinguish these organophosphates from those that do not cause delayed neuropathy in animal models.

Animal Testing Alternatives↗

PubMed1995

Esterase comparison in neuroblastoma cells of human and rodent origin.

1. Activities of acetylcholinesterase (AChE), neuropathy target esterase (NTE), and carboxylesterase (CbxE) were compared in neuroblastoma cells of human origin (SH-SY5Y) and murine origin (NB41A3). 2. Mouse neuroblastoma cells had lower specific activities of NTE and CbxE than did human neuroblastoma cells; specific activities in the murine cells correlated with specific activities in mouse brain. 3. AChE activities in mouse and human neuroblastoma cells were considerably lower than AChE activities in mouse or hen brain. 4. Inhibition of esterases did not demonstrate interspecies differences for 12 of the 17 anti-esterase compounds tested with human and mouse neuroblastoma cells.

Acetylcholinesterase↗

PubMed1994

Interaction of organophosphorus compounds with muscarinic receptors in SH-SY5Y human neuroblastoma cells.

Human neuroblastoma cells (line SH-SY5Y) were used to examine the interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH-SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phosphorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and mipafox (N,N'-diisopropyl phosphorodiamide fluoridate) that ranged between 10(-9) M and 10(-3) M (10(-2) M for mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]-N-methylscopolamine (3H-NMS). Concentrations of paraoxon > 0.5 x 10(-3) M and PSP 1 x 10(-3) M significantly inhibited the binding of a saturating concentration of 3H-NMS. Concentrations of > 10(-5) M paraoxon or PSP could significantly inhibit the binding of a half-saturating concentration of 3H-NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX-116 for M2, and 4-DAMP for M3) indicated that SH-SY5Y cells have muscarinic receptors most sensitive to the specific antagonist for the M3 subtype (IC50 of 10(-8) M for 4-DAMP compared to 2.5 x 10(-6) M and 2.7 x 10(-5) M for pirenzepine and AFDX-116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phosphoinositides the capability of OPs to alter levels of inositol phosphates and agonist-stimulated increases in inositol phosphate formation was examined. Intact cells were prelabeled with [3H]myo-inositol and then incubated for 15 min with the OPs before addition of 10(-5) M to 10(-3) M carbachol. Levels of inositol phosphates were determined as the amount of aqueous soluble radiolabeled product extracted from the reaction mixture. Paraoxon and PSP, but not mipafox or DFP, decreased basal levels of inositol phosphates in a concentration-related manner. This could be overcome in cells stimulated with carbachol, a muscarinic agonist, and with sodium fluoride, which does not act at muscarinic receptors. These results indicate that certain OPs, upon acute exposure, interact with muscarinic receptors, but that they also have effects on levels of inositol phosphates that may be associated with another site of action in SH-SY5Y cells.

Carbachol↗

PubMed1994

Neuropathy target esterase inhibition by organophosphorus esters in human neuroblastoma cells.

Certain organophosphorus compounds (OPs) produce a delayed neuropathy (OPIDN) in man and some animal species. Capability to cause OPIDN is generally predicted in animal models by early and irreversible inhibition of neuropathy target esterase (NTE, neurotoxic esterase). In this study, NTE inhibition in response to OP exposure was examined in cell culture, using the human SH-SY5Y neuroblastoma cell line. Cells were exposed for 1 hr to equimolar (1 x 10(-5) M) concentrations of 6 OPs associated with OPIDN in vivo (including 2 protoxicants and 4 active (-P = O) toxicants), and 8 OPs that do not produce delayed neuropathy in animal models (including 5 protoxicants and 3 -P = O compounds). The -P = O compounds that cause OPIDN in animal models inhibited NTE > 60% at the test concentration; -P = O compounds that do not cause OPIDN in animal models inhibited NTE < 30%. Protoxicants did not inhibit NTE at the test concentration, reflecting their limited metabolism in the human cell line. These results indicate that human neuroblastoma cells have potential use in the initial screening of bioactive OPs with capability for causing OPIDN.

Carboxylic Ester Hydrolases↗