Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. / Ph. D.

Coumaphos is an organophosphate insecticide used predominantly by the US Department of Agriculture, Animal and Plant Health Inspection Services for its tick eradication program. Bioremediation of the hydrolysis products of coumaphos, chlorferon and diethylthiophosphate (DETP), using Ca-alginate immobilized cells was the focus of this research. Consortia of indigenous microorganisms capable of degrading chlorferon and DETP were isolated separately. Since chlorferon inhibited both chlorferon-degrading and DETP-degrading organisms, it was not possible to enrich a consortium of organisms for simultaneous degradation of chlorferon and DETP. A two-step growth procedure was developed for degradation studies to provide biomass acclimated to the target compound and reaction medium since cells lost their degradation activity during the growth in a rich medium. Without acclimation, approximately a week-long lag period was required before degradation was initiated. Optimum reaction conditions were found for the degradation of chlorferon and DETP using free cells. Reaction kinetics of chlorferon and DETP were determined using enzyme kinetics because cell growth was not observed during the degradation. Chlorferon degradation followed substrate inhibition kinetics and DETP degradation followed simple Michaelis-Menten kinetics. A calcium-alginate immobilized cell system was developed, and the optimum bead loadings in the reactor were determined. Degradation rates for immobilized cells were enhanced up to five times that for free cells in untreated cattle dip (UCD) solution. The enhanced degradation of immobilized cells was due to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of cells caused by Ca-alginate immobilization may have contributed to a slightly increased reaction rate in pure solution. Diffusion coefficients of chlorferon and DETP into Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients of chlorferon and DETP increased with increasing agitation speed and decreasing substrate concentration. Increased cell concentration in gel beads caused lower diffusivity. Calcium-alginate gel beads used in this study were not subject to diffusional limitations. Both external and internal mass transfer resistances were negligible, and the degradation rate inside Ca-alginate gel beads was reaction-limited.

The United States Environmental Agency permits the use of 39 organophosphate pesticides. Many of these pesticides are acutely toxic and have lasting effect on human health. Organophosphates quickly metabolize in the body, therefore currently human exposure is studied by measuring the metabolic products in urine. In this work a suite of analytical methods was developed to determine the presence of un-metabolized organophosphate pesticides in human serum. First mass spectroscopic detection methods were evaluated. Gas chromatograph coupled tandem mass spectrometer was used to compare the detection limits using chemical and electron impact ionization. Positive chemical ionization was selected, because it provided better detection limits for this set of analytes. Liquid chromatograph coupled tandem mass spectrometry was also evaluated and was found advantageous over the gas chromatographic method for approximately 50% of the compounds. Positive atmospheric pressure chemical ionization was chosen for this group of compounds. Once the analytes were separated by detection methods, analytical separation methods were compared: column and eluent was selected for liquid chromatography, column alone was selected for gas chromatography. Last step of the method development was to produce a suitable sample cleanup process. Solid phase extraction was not suitable because the very wide range of solubility characteristics and hydrolytic stability of the analytes. Lyophilization, liquid-liquid extraction methods were tested and compared. A multi step cleanup method was produced, which starts with liquid-liquid extraction using high pressure ethyl acetate in accelerated solvent extractor, solvent exchange and a lipid removal step. The concentrated extract then injected in a HPLC-MS-MS system then the same extract either directly injected in GC-MS-MS or further purified using headspace solid phase micro extraction before the GC-NS-MS step. The method was used with good results for analyzing samples collected from farm workers using OP pesticides.

Coumaphos is an organophosphate insecticide used predominantly by the US Department of Agriculture, Animal and Plant Health Inspection Services for its tick eradication program. Bioremediation of the hydrolysis products of coumaphos, chlorferon and diethylthiophosphate (DETP), using Ca-alginate immobilized cells was the focus of this research. Consortia of indigenous microorganisms capable of degrading chlorferon and DETP were isolated separately. Since chlorferon inhibited both chlorferon-degrading and DETP-degrading organisms, it was not possible to enrich a consortium of organisms for simultaneous degradation of chlorferon and DETP. A two-step growth procedure was developed for degradation studies to provide biomass acclimated to the target compound and reaction medium since cells lost their degradation activity during the growth in a rich medium. Without acclimation, approximately a week-long lag period was required before degradation was initiated. Optimum reaction conditions were found for the degradation of chlorferon and DETP using free cells. Reaction kinetics of chlorferon and DETP were determined using enzyme kinetics because cell growth was not observed during the degradation. Chlorferon degradation followed substrate inhibition kinetics and DETP degradation followed simple Michaelis-Menten kinetics. A calcium-alginate immobilized cell system was developed, and the optimum bead loadings in the reactor were determined. Degradation rates for immobilized cells were enhanced up to five times that for free cells in untreated cattle dip (UCD) solution. The enhanced degradation of immobilized cells was due to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of cells caused by Ca-alginate immobilization may have contributed to a slightly increased reaction rate in pure solution. Diffusion coefficients of chlorferon and DETP into Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients of chlorferon and DETP increased with increasing agitation speed and decreasing substrate concentration. Increased cell concentration in gel beads caused lower diffusivity. Calcium-alginate gel beads used in this study were not subject to diffusional limitations. Both external and internal mass transfer resistances were negligible, and the degradation rate inside Ca-alginate gel beads was reaction-limited.

The aim of this research was to determine whether a series of novel pyridinium oximes that were synthesized to incorporate phenoxyalkyl moieties to increase lipophilicity, and thereby increase their likelihood of crossing the blood-brain barrier, could effectively reactivate phosphorylated AChE in vitro. An experimental OP was synthesized as a surrogate for sarin to test the reactivation potential of the oximes, phthalimidyl isopropyl methylphosphonate (PIMP). The reactivation activities of the novel pyridinium oximes on PIMP exposed AChE and structure activity relationships were examined. Differences in reactivation potential in comparison to the widely used 2-PAM were also examined. All the novel oximes tested demonstrated some ability to reactivate inhibited AChE. Percent reactivations varied among the oximes (24%-78%), and the novel oximes were not as effective as 2-PAM or TMB-4, which reactivated 91 and 97%, respectively. The lipophilicity for all oximes was greater than 2-PAM or TMB-4 by 3- to 374- fold. A few of the novel oximes showed combined higher lipophilicity and reactivation potential approaching that of 2-PAM, and therefore suggest some potential efficacy as brain-penetrating oxime reactivators.

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of organophosphorus compounds. Metal ion roles during binding and catalysis are probed by comparing the kinetic properties of Zn/Zn, Cd/Cd, and Zn/Cd PTE with a variety of phosphate trisesters. The metal in the α-site of the binuclear metal center modulates the pKa values determined from pH-rate profiles. These results suggest that the α-metal is responsible for activating the nucleophilic hydroxide. In an effort to determine the function of the β-metal, the kinetic parameters for diethyl p-chlorophenyl thiophosphate are compared with diethyl p-chlorophenyl phosphate. The thiophosphate substrate is hydrolyzed 20 to 100-times faster than the phosphate substrate for Zn/Zn, Cd/Cd, and Zn/Cd PTE. When Cd2+ occupies the β-site, the inverse thio effect increases which suggests polarization by the β-metal on the phosphoryl oxygen or sulfur bond. The catalytic roles of Asp 233, His 254, and Asp 301 are determined by comparing the kinetic parameters of a series of alanine and asparagine mutations with paraoxon and diethyl p-chlorophenyl phosphate. The increased rate of hydrolysis for diethyl p-chlorophenyl phosphate with the mutants is consistent with the existence of a proton relay system from Asp 301 to His 254 to Asp 233. A detailed mechanism for the hydrolysis of organophosphates by PTE has been proposed. PTE hydrolyzes a number of chiral organophosphorus esters. The pKa of the leaving group phenol is altered for a series of chiral phosphate, phosphonate, and phosphinate esters. The stereoselectivity of wild-type, G60A, and I106G/F132G/H257Y PTE is enhanced as the pKa value of the leaving group phenol increases for phosphate, phosphonate, and phosphinate esters. In addition to improving the stereoselectivity of phosphotriesterase, mutations that affect the size of the active site of PTE are screened to identify a mutant enzyme that preferentially hydrolyzes the opposite isomer of wild-type PTE. The rate constants and stereoselectivity ratios for a number of active site mutants have been determined. H254Y/L303T PTE reverses the stereoselective preference of phosphonate and phosphinate substrates. The PTE stereoselectivity of O-methyl, O-phenyl acetylphenyl phosphate is reversed 970-fold by I106G/F132G/H257Y. A reversal mutant was resolved for phosphate, phosphonate, and phosphinate esters.

No description available.

Organophosphates (OPs), including nerve agents, target the cholinergic system via inhibition of acetylcholinesterase (AChE), with subsequent overstimulation resulting in neural damage and potential detrimental long-term effects. The efficacy of novel pyridinium oxime reactivators, created with moieties to increase blood-brain barrier penetration, was tested using highly relevant sarin and VX surrogates. Glial fibrillary acidic protein (GFAP; an indicator of neural damage) and monoamines (dopamine, serotonin, and their metabolites) were measured in select brain regions via immunohistochemistry and HPLC, respectively. Adult male rats were treated ip with high, sub-lethal doses of surrogates for sarin or VX, nitrophenyl isopropyl methylphosphonate (NIMP) or nitrophenyl ethyl methylphosphonate (NEMP), respectively. Surrogate treatment was followed after 1 hr by im administration of novel oxime. Seizure activity was monitored, and kainic acid (KA) served as a positive control. Administration of KA or surrogate (NIMP or NEMP) significantly increased GFAP expression compared to control animals. Two different formulations of one particular oxime (bromide vs. mesylate salt) attenuated seizures and reduced GFAP levels over NIMP or NEMP treatments alone to levels near those of controls in both the piriform cortex and dentate gyrus region of the hippocampus, while 2-PAM did not provide protection. Serotonergic activity was increased in several brain regions, including the piriform cortex, one hr after NIMP treatment. Markers of oxidative stress (isoprostanes) were also tested. Overall, these results indicate the potential therapeutic efficacy of these oximes and suggest this innovative chemistry may protect against neural damage induced by OP.

Previous monitoring studies by federal agencies such as the United States Geological Survey have shown that environmental contaminants rarely occur as single compounds but, rather, as mixtures. In aquatic ecosystems, mixtures of these compounds are often complex, sometimes containing dozens of compounds across a number of different chemical classes. Non-target aquatic organisms are frequently exposed to varying levels of contaminants based upon the physical properties of the chemicals, such as water solubility, and life-cycle habits of the individual organisms. In addition to this, past research has indicated that the presence of one class of contaminant may have an influence on the toxicities of other chemical classes. Water-only toxicity testing has historically provided a means by which researchers can rapidly determine the toxic effects of water-soluble compounds such as triazine herbicides and organophosphate insecticides. However, many legacy pesticides, such as organochlorine, and some current-use pesticides, such as pyrethroids, are strongly hydrophobic, and suspended or bedded sediments, rather than water, would generally be more appropriate matrices for monitoring. Yet sampling of sediments and quantification of residues of these pesticides is often lacking. Similarly, there have been few studies examining the toxicity of mixtures of these compounds in sediment. The first goal of this research was to examine the effects of select triazine herbicides on organophosphate insecticide toxicity utilizing water-only toxicity test with the aquatic amphipod Hyalella azteca. The second goal was to analyze an existing database of chemical concentrations using a toxicity-based screening approach in order to estimate the environmental hazard posed by mixtures of pyrethroid, organochlorine, and organophosphate insecticides in sediment to H. azteca. The third goal of this research was to examine the toxic effects of mixtures of different pyrethroid insecticides to H. azteca using compounds identified as most relevant from the screening phase of the study. The fourth goal of this research was to examine how pyrethroid and organochlorine insecticides partition between different size fractions within sediment and detritus, as well as between sediments with differing organic carbon content, and the resulting effects to compound toxicity and bioavailability. The final goal of this research was to examine potential modifications to bifenthrin sediment partitioning, toxicity, and bioaccessibility resulting from various dissolved salt concentrations in overlying water using H. azteca and Chironomus dilutus as reference organisms. Together, the individual objectives of this study provide a thorough and multi-tiered approach to determining the occurrence, environmental fate, biological effects, and bioavailability of frequently detected and co-occurring environmental contaminants in both agricultural and urban landscapes.

No description available.