Grantee Research Project Results
Final Report: Refining an Alternative to the EPA's Hen Test for OPs
EPA Grant Number: R825356Title: Refining an Alternative to the EPA's Hen Test for OPs
Investigators: Ehrich, Marion , Jortner, Bernard S.
Institution: Virginia Tech
EPA Project Officer: Aja, Hayley
Project Period: November 7, 1996 through November 6, 1999 (Extended to November 6, 2000)
Project Amount: $357,572
RFA: Exploratory Research - Human Health (1996) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The current EPA-sanctioned test for screening of organophosphorus (OP) insecticides involves dosing of hens, collection of brain and spinal cord for neurotoxic esterase (NTE) and acetylcholinesterase (AChE) determinations, and perfusion-fixation of peripheral nerve and spinal cord for morphological indices of organophosphorus-induced delayed neuropathy (OPIDN) which follows NTE inhibition. Previous work indicated that the biochemical endpoints could be detected in neuroblastoma cells as long as active toxicants were used, and that the ratio of NTE to AChE inhibition could identify OP compounds capable of causing OPIDN. The specific objectives of this project are to: (1) develop a means for activation of phosphorothionate protoxicants so they will inhibit esterases in vitro in human neuroblastoma cells using inorganic activation (bromine solution) and noncytotoxic microsomal enzyme systems; (2) examine esterase inhibition following multiple dosing procedures of OP compounds in human neuroblastoma cells; and (3) explore development of an in vitro morphological correlates for OPIDN in human neuroblastoma cells and chick reaggregate cultures.Summary/Accomplishments (Outputs/Outcomes):
Biochemical studies in SH-SY5Y human neuroblastoma cells demonstrated that active esterase inhibitors could be produced from the OP protoxicants parathion, chlorpyrifos, leptophos, EPN, malathion and fenitrothion. This could be accomplished by oxidation in the presence of bromine as catalyst and also by incubation with rat liver microsomes. Bromine produces only the esterase-inhibiting oxon; oxons and other metabolites were formed by the rat liver microsomes. For this reason, inhibitions of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) were usually greater when bromine was used to activate the organophosphate protoxicant. Tri-ortho-tolyl phosphate (TOTP), which requires more than oxidation for activation, was not covered to an esterase-inhibiting product by bromine, but activation did occur with rat liver microsomes. Studies with human microsomes indicated that CYP450 3A4 was an isozyme of primary importance in the activation of chlorpyrifos-oxon.Multiple-dose (28-day) studies with low concentrations (10-11 ? 10-7 M) chlorpyrifos and chlorpyrifos-oxon, TOTP and PSP, and parathion and paraoxon in serum-free media indicated that these concentrations had effects on AChE that were not seen with single exposures. Sensitivity of NTE was not markedly increased, nor was there increased sensitivity to challenge by chlorpyrifos-oxon.
Morphological investigations at the light microscopic and ultrastructural level suggest that the osmotic environment of neurons in chick reaggregate cultures could be disrupted by chlorpyrifos-oxon and that intracytoplasmic cisternal hyperplasia appeared after exposure to phenyl saligenin phosphate (PSP), an OP compound that causes delayed neuropathy. The latter lesion also is seen in hens with OPIDN. Parathion and paraoxon, which are cholinesterase-inhibiting rather than neuropathy-inducing OP compounds, appeared to increase cytoplasmic clearing in human neuroblastoma cells.
Mechanistic studies on OPIDN included calpain activation and CaMg-ATPase inhibition. Calpain expression in hen lumbar spinal cord, which contains the cell bodies of neurons that innervate the hind limbs, was increased 2 days after treatment with 2.5 mg/kg PSP. Calpain can also be detected in chick reaggregate cultures. CaMg-ATPase in brain synaptosomes was inhibited after in vivo and in vitro treatment with certain OP compounds. However, inhibition did not occur with all neuropathic OP compounds, indicating that inhibition of this enzyme is not necessary for development of OPIDN.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
| Other project views: | All 20 publications | 4 publications in selected types | All 4 journal articles |
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Barber D, Correll L, Ehrich M. Comparative effectiveness of organophosphorus protoxicant activating systems in neuroblastoma cells and brain homogenates. Journal of Toxicology and Environmental Health 1999;57(1):63-74 |
R825356 (1999) R825356 (Final) |
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Barber D, Correll L, Ehrich M. Comparison of two in vitro activation systems for protoxicant organophosphorous esterase inhibitors. Toxicological Sciences 1999;47:16-22. |
R825356 (1999) R825356 (Final) |
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Barber D, Hunt J, Ehrich M. Inhibition of calcium-stimulated ATPase in the hen brain P2 synaptosomal fraction by organophosphorus esters: relevance to delayed neuropathy. Journal of Toxicology and Environmental Health, Part A 2010;63(2):101-113 |
R825356 (1999) R825356 (Final) |
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Tiffany-Castiglioni E, Ehrich M, Dees L, Costa LG, Kodavanti PRS, Lasley SM, et al. Bridging the gap between in vitro and in vivo models for neurotoxicology. Toxicological Sciences 1999;51(2):178-183 |
R825356 (1999) R825356 (Final) |
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Supplemental Keywords:
organophosphorus compounds, delayed neuropathy, in vitro alternative., Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, ecological risk assessment, neurotoxic, multiple acute exposure, neuroblastoma cells, organophosphorous, animal model, developmental effects, human exposure, NTE, EPA's hen test, biomarker, cell stress proteinsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.