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.

The serine/cysteine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) has been used both to promote and to protect against neuropathic events of organophosphorus-induced delayed neuropathy (OPIDN) in hens (Lotti et al., 1991; Veronesi et al., 1985; Pope and Padilla, 1990; Pope et al., 1993). This study expands upon this work by correlating clinical and neuropathological findings in these modifications of OPIDN. To provide appropriate models of OPIDN, single phenyl saligenin phosphate (PSP) dosages of 0.5, 1.0, or 2.5 mg/kg were administered to adult hens. PMSF (90 mg/kg) was given either 4 hours after or 12 hours prior to PSP administration. Clinical signs and pathologic changes in the biventer cervicis nerve (El-Fawal et al., 1988) were monitored. PSP alone, 2.5 mg/kg, ellicitated severe OPIDN (terminal clinical score 7.5 & ± 1.0 [0-8 scale]; neuropathology score 2.7 ± 0.3 [0-4 scale, based on myelinated fiber degeneration]). PMSF given 12 hours prior to PSP gave complete protection (clinical and neuropathology scores of 0; p<0.0001). Signs and lesions of OPIDN were absent following 0.5 mg/kg PSP alone, but PMSF given 4 hours after PSP potentiated its neurotoxic effects (clinical score 4.0 ± 0.0; neuropathology score 3.5 ± 0.3; p<0.0001). At the time of sacrifice, there was a correlation (r = 0.61) between the clinical score on the last day of observation and the neuropathology scores (p<0.0001). This study demonstrates that the intensity of peripheral nerve myelinated fiber degeneration correlates with clinical deficits in PMSF-induced potentiation and protection in OPIDN. / Master of Science