Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1954. / Typescript. Vita. Includes reprints of various journal articles by the author et al. Includes bibliographical references (leaves 116-136).

The organophosphate resistance-associated elevated esterases Estα2, Estβ1 and Estβ2 were purified to homogeneity from larvae of the Cuban Habana strain. The bimolecular rate constants (kas) of Habana Estβ1 with a range of organophosphates were not significantly different to those of PelRR Estβ21 , and were higher with some organophosphates than PelRR Estα21 (Karunaratne et al, 1993). The relative insecticide binding efficiency of these esterases could not, therefore, explain why co-amplified estα2 and estβ2 are out competing estβ1 in the field. On the basis of their kas, both Habana Estα2 and Estβ2 could be distinguished from their equivalents purified from other strains. In two organophosphate resistant strains of Culex quinquefasciatus from Colombia and Trinidad, possessing the amplified esterase genes estα3 and estβl, the EcoRI restriction fragment lengths of the estβl genes and their flanking regions were different both to each other and to those previously reported for TEM-R estβ11 (Raymond et aL, 1991) and MRES estβ12 (Vaughan et aL, 1995). There were a number of significant differences between the kas of purified Colombia, Trinidad and Habana Estβ1s. The low kas and high k3s for the interaction of Colombia Estβ1 with several insecticides confirmed that, as for Estα21 and Estβ21, the main role of Estβ1 is sequestration. The kas of Habana, Colombia and Trinidad Estβ1s were higher than that of the electrophoretically identical Est'β13 purified from the susceptible PelSS strain (Karunaratne et al, 1995a). This suggests that the elevated esterase-based mechanism confers resistance through amplification of alleles coding C for esterases having a higher reactivity with the insecticides they sequester than esterases coded for by their non-amplified counterparts. A PelRR Estα21 antiserum had the same cross-reactivity with Habana Estα2 as with Estα21. However, both Habana Estβ1 and Estβ2 had a cross-reactivity of approximately 150-fold less than the Estα2s.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 64-68).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 54-61).

Phosphorothioate insecticides such as parathion (0,0, diethyl O-pnitrophenyl phosphorothioate) must undergo metabolic activation to form oxygen analogs in order to exert their toxicity. The specific isoforms of cytochrome P450 involved in this oxidative desulphuration reaction were examined in rat liver microsomes and a panel of sixteen human liver microsomes. In the rat, parathion (20μM and 200μM) was activated to paraoxon with an apparent Km of 10.4±0.25μM (n=3), the metabolic rates were 241±17 and 256±18 pmol/min/mg protein, respectively. p-Nitrophenol was also formed, at 235±15 and 220±23 pmol/min/mg protein. Human liver microsomes activated parathion (20μM and 200μM) with an apparent Km of 9μM-16μM (n=3), the metabolic rates were 23.3-199.3 and 18.7-310.3 pmol/min/mg protein (n=16). p-Nitrophenol was also formed, at 321.1- 769.2 and 406.2-778.3 pmol/min/mg protein. The activation of parathion (200μM) by human liver microsomes was positively correlated with nifedipine oxidation, indicating the involvement of CYP3A. Correlations were not significant with ethoxyresorufin-0-dealkylation, pentoxyresorufin-0-dealkylation, pnitrophenol hydroxylation, paraoxon hydrolysis or phenylvalerate hydrolysis. Paraoxon formation from parathion by human liver microsomes was markedly inhibited by the CYP3A inhibitors ketoconazole, quercetin and naringenin (apparent Ki=21μM). Metyrapone and a-naphthflavone had some inhibitory effect. The inhibitors were generally less effective towards parathion metabolism by rat liver microsomes. Experiments with EDTA indicated that A-esterase was not functionally important at low levels of paraoxon. Human P450s 3A4 and 3A5 expressed microsomes were the most efficient at biotransforming parathion to paraoxon, although P450s 1A1,2B6 and 2C8 also catalysed the reaction. The present study has shown marked interindividual variation in the metabolism of parathion, which may influence toxicity following exposure to this or other phosphorothioates. Co-administration of inhibitors or inducers of the enzymes involved may affect the fate of parathion and thus enhance or reduce its toxicity.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1953. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 56-58).

The mechanism of toxic action for organophosphates (OPs), originally developed as insecticides, is the persistent inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and subsequent hyperstimulation of the nervous system. Many OPs require bioactivation via cytochromes P450 to oxon metabolites which are anticholinesterases. Organophosphates display a wide range of acute toxicities. Differences in the OPs’ chemistries results in differences in the compounds' 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. 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: 1) age-related sensitivity of AChE, BChE and CaE to structurally different OPs in rat tissues; 2) interspecies and intraspecies differences in bioactivation and detoxication of the OP insecticide malathion in rat and human hepatic microsomes; and 3) interspecies and intraspecies differences in sensitivity of AChE from erythrocyte ghost preparations to malaoxon. Sensitivities of esterases to 12 OPs was assessed by IC50s. The OPs displayed a wide range of AChE IC50s (low nM-µM) with no differences among ages; however, the CaE IC50s generally increased with age (up to 100old) reflecting greater protection in adults. Kinetic analysis of the bioactivation of malathion to the anticholinesterase metabolite, malaoxon, was measured in hepatic microsomes from rats (adult) and humans (various ages) of both sexes. No statistical interspecies (rat and human) or intraspecies (among humans) differences were found. The CaE degradation of malathion and malaoxon was determined in the microsomal samples using indirect measurements. No interspecies or intraspecies differences were found; however, CaE activity in rat microsomes was significantly higher than in humans. Inhibition of AChE by malaoxon was analyzed kinetically in erythrocyte ghost preparations from rats (adults) and humans (three age groups) of both sexes. No statistical interspecies or intraspecies differences were found. These results suggest the age-related differences in acute toxicities of OPs in mammals is primarily a result of their detoxication capacity.

No description available.

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.