Organochlorine pesticides (OCPs) were extensively applied in agriculture, industry and public health programs for decades. Based on the persistence and the lipophilicity of OCPs, these chemicals are ubiquitous in the environment and can be accumulated in fatty tissues of animals through the food chain. Even being restricted for years, OCPs are still detected in human bodies. In this thesis, analytical methods for the determination of OCPs were developed and applied for the analysis of cord serum samples. The evaluation of prenatal exposure to OCPs and its effects on birth outcomes as well as the postnatal growth were investigated. Due to the toxicology and carcinogenesis, biomonitoring of the OCP exposure to human is needed. Therefore, an analytical method with high sensitivity and specificity is required to detect OCPs at trace levels in serum. We compared two data acquisition modes of mass spectrometry (MS), namely selected ion monitoring (SIM) and multiple reaction monitoring (MRM). Higher sensitivity and selectivity were achieved by MRM because the background noise was reduced by lowering the matrix effects. Different ionization techniques, including electron ionization (EI), chemical ionization (CI) and atmospheric pressure chemical ionization (APCI) were evaluated. The EI source is a universal ionization technique available with the MS library for the compound identification. The negative chemical ionization (NCI) is more suitable to analyze compounds with high electronegativity. The novel ionization technique APCI was coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS). The APCI source was evaluated by terms of ionization and fragmentation performance. APCI was a soft ionization technique generating molecular ions with high intensity. The selectivity and sensitivity of APCI were comparable or better than the EI source. As one of the largest consumers and producers of OCPs, China has suffered severe OCP pollution. Previous monitoring studies reported detectable levels of OCPs in human bodies. However, studies concerning the prenatal exposure to OCPs in China are limited. Due to the vulnerability of fetuses, the effects of prenatal exposure to OCPs could be more severe than those of adults. We collected cord serum samples during the delivery period in Wuhan, China and measured the OCP concentrations to assess the prenatal exposure by using GC-MS/MS. Compared with other areas in China, the OCP levels in Wuhan were comparable in this population. The identified predominant OCPs were β-HCH and p,p'- DDE, with geometric means of 8.67 and 33.9 ng/g lipid, respectively. Slight positive associations were found between α-HCH and β-HCH, and between o,p′- DDT and p,p′-DDT, which indicated similar exposure source of these chemicals. The obtained results showed that HCH levels were associated with maternal age, body mass index (BMI) before pregnancy, education levels, and passive smoking. Associations between the prenatal exposure of OCPs and birth outcomes were investigated. The sex-specific relationships between the OCP exposure and birth size were indicated. Concentrations of β-HCH were inversely associated with birth weight and ponderal index for boys, while for girls these associations were not significant. Our results suggested that the prenatal exposure to OCPs exerted negative effects on the fetal growth, and precautions should be taken even though the OCP levels were relatively low.

The effect of the following pesticides on DC electrical conductivity of lecithin-cholesterol membranes has been studied: endothall, paraquat, diquat, 2,4-D, 2,4-DB, 2,4,5-T, 2,4-DCP. It has been found that the ions of endothall, paraquat and diquat are essentially membrane impermeable and that they do not bind to the membrane surface. In contrast, 2,4-D, 2,4-DB, 2,4,5-T and 2,4-DCP induce electrical conductivity in lecithincholesterol membranes and in addition they also cause an increase in the nonactin-K+ membrane conductivity. The compounds 2,4-D, 2,4-DB, 2,4,5-T and 2,4-DCP basically behave as class II uncouplers. The kinetic scheme of charge transfer across the membrane, based on the assumption that the membrane is permeable both to the negatively charged dimers and to the neutral molecules of pesticides, satisfactorily explains the basic features of the experimental results: the concentration dependence of pesticide-induced membrane conductance, effect of proton concentration on membrane conductance, and the effect of pesticide concentration on the voltage dependence of membrane conductance. It fails to predict the effect of proton concentration on the voltage dependence of membrane conductance. The enhancement of nonactin-K+ membrane conductance by the pesticide is presumably due to the adsorption of the ionized form of the pesticide at the membrane surface. It was found that the Gouy-Chapman diffuse double layer theory was not applicable for the calculation of surface membrane potential due to the adsorbed ions.

Chow, Wing Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 118-129). / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xv / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Pesticide contaminations in the environment --- p.1 / Chapter 1.2 --- Pesticides --- p.1 / Chapter 1.2.1 --- Usage of pesticide in the world --- p.1 / Chapter 1.2.2 --- Organochlorine (OC) pesticides --- p.3 / Chapter 1.2.3 --- Organophosphate (OP) pesticides --- p.4 / Chapter 1.2.4 --- Carbamate pesticides: --- p.6 / Chapter 1.2.5 --- Pyrethroid pesticides: --- p.6 / Chapter 1.3 --- Toxicological model: Zebrafish --- p.7 / Chapter 1.4 --- Biomarkers --- p.9 / Chapter 1.4.1 --- Cytochrome P450 1A (CYP1A) --- p.12 / Chapter 1.4.2 --- Cytochrome P450 3A65 (CYP3A65) --- p.14 / Chapter 1.4.3 --- Biomarker for estrogenicity - Vitellogenin (VTG1) --- p.15 / Chapter 1.4.4 --- Catalase (CAT) and Glutathione S-transferase (GST) --- p.18 / Chapter 1.4.4.1 --- Catalase (CAT) --- p.18 / Chapter 1.4.4.2 --- Glutathion S-transferase (GST) --- p.19 / Chapter 1.4.5 --- Multiple Drug Resistance (MDR1) --- p.20 / Chapter 1.4.6 --- Acetylcholinesterase (AChE) --- p.21 / Chapter 1.5 --- Objectives of this study --- p.26 / Chapter Chapter 2 --- "Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to organochlorine pesticides: endosulfan, heptachlor and methoxychlor" --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and methods --- p.30 / Chapter 2.2.1 --- Chemicals tested --- p.30 / Chapter 2.2.2 --- Zebrafish cultivation and egg production --- p.30 / Chapter 2.2.3 --- Determination of 96h-EC50 and 96h-LC50 of organochlorine pesticides and bisphenol-A for zebrafish embryo-larvae --- p.31 / Chapter 2.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.31 / Chapter 2.2.5 --- Extraction of total RNA from the exposed embryo-larvae samples --- p.32 / Chapter 2.2.6 --- Reverse Transcription --- p.33 / Chapter 2.2.7 --- Quantifications of mRNA levels by qPCR --- p.35 / Chapter 2.2.7.1 --- Primer design --- p.35 / Chapter 2.2.7.2 --- Validation of qPCR conditions --- p.36 / Chapter 2.2.7.3 --- Quantification of biomarker gene expression levels in zebrafish embryo-larvae --- p.42 / Chapter 2.2.8 --- Statistical analysis --- p.43 / Chapter 2.3. --- Results --- p.44 / Chapter 2.3.1 --- Toxicities of OC pesticides and bisphenol-A --- p.44 / Chapter 2.3.2 --- Effects of OC pesticides and bisphenol-A on biomarker gene expression levels --- p.44 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1 --- Toxicities of OC pesticides and bisphenol-A --- p.60 / Chapter 2.4.2 --- Effects of OC pesticides on CYP1A gene expression --- p.61 / Chapter 2.4.3 --- Effects of OC pesticides on CYP3A65 gene expression --- p.61 / Chapter 2.4.4 --- Effects of OC pesticides on VTG1 gene expression --- p.63 / Chapter 2.4.5 --- Effects of OC pesticides on MDR1 gene expression --- p.64 / Chapter 2.5 --- Conclusion --- p.65 / Chapter Chapter 3 --- "Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to a organochlorine pesticide, chlorpyrifos" --- p.66 / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.2 --- Materials and methods --- p.68 / Chapter 3.2.1 --- Chemicals tested --- p.68 / Chapter 3.2.2 --- Zebrafish cultivation and egg production --- p.68 / Chapter 3.2.3 --- Determination of 96h-EC50 and 96h-LC50 of chlorpyrifos for zebrafish embryo-larvae --- p.68 / Chapter 3.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.68 / Chapter 3.2.5 --- Extraction of total RNA from the exposed embryo-larvae samples --- p.69 / Chapter 3.2.6 --- Reverse Transcription --- p.69 / Chapter 3.2.7 --- Quantifications of mRNA levels by qPCR --- p.70 / Chapter 3.2.7.1 --- Primer design --- p.70 / Chapter 3.2.7.2 --- Validation of qPCR conditions --- p.70 / Chapter 3.2.7.3 --- Quantification of biomarker gene expression levels in zebrafish embryo-larvae --- p.75 / Chapter 3.2.8 --- Determination of acetylcholinesterase (AChE) activities --- p.76 / Chapter 3.2.9 --- Statistical analysis --- p.77 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Toxicities of chlorpyrifos --- p.78 / Chapter 3.3.2 --- Effects of chlorpyrifos on CAT and GST gene expression levels --- p.81 / Chapter 3.3.3 --- Effects of chlorpyrifos on acetylcholinesterase (AChE) activity --- p.83 / Chapter 3.4 --- Discussions --- p.86 / Chapter 3.4.1 --- Toxicity of chlorpyrifos --- p.86 / Chapter 3.4.2 --- Effect of chlorpyrifos on CAT and GST gene expressions --- p.86 / Chapter 3.4.3 --- Effect of chlorpyrifos on AChE activity --- p.88 / Chapter 3.5 --- Conclusions --- p.89 / Chapter Chapter 4 --- Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to carbamate and pyrethroid pesticides --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.2 --- Materials and methods --- p.92 / Chapter 4.2.1 --- Chemicals tested --- p.92 / Chapter 4.2.2 --- Zebrafish cultivation and egg production --- p.92 / Chapter 4.2.3 --- Determination of 96h-EC50 and 96h-LC50 of aldicarb and cypermethrin for zebrafish embryo-larvae --- p.92 / Chapter 4.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.92 / Chapter 4.2.5 --- Quantification of biomarker gene expression levels in zebrafish embryo- larvae and Determination of acetylcholinesterase (AChE) activity --- p.94 / Chapter 4.2.6 --- Statistical analysis --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Toxicities of aldicarb and cypermethrin --- p.95 / Chapter 4.3.2 --- Effects of aldicarb and cypermethrin on CAT and GST gene expression levels.. --- p.99 / Chapter 4.3.3 --- Effects of aldicarb on acetylcholinesterase (AChE) activity --- p.102 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Toxicity of aldicarb of cypermethrin --- p.105 / Chapter 4.4.2 --- Effect of aldicarb and cypermethrin on CAT and GST gene expressions --- p.105 / Chapter 4.4.3 --- Effect of aldicarb on AChE activity --- p.107 / Chapter 4.5 --- Conclusion --- p.108 / Chapter Chapter 5 --- General Conclusion --- p.109 / Chapter 5.1 --- Toxicities of pesticides --- p.109 / Chapter 5.2 --- Effects of OC pesticides on biomarker gene expressions --- p.113 / Chapter 5.3 --- "Effects of chlorpyrifos, aldicarb and cypermetrhin on biomarker gene expressions" --- p.116 / Chapter 5.4 --- Effect of chlorpyrifos and aldicarb on AChE activity --- p.116 / References --- p.118

Orientador: Luiz Antônio de Lima Resende / Banca: Cláudia Ferreira da Rosa Sobreira / Banca: José Luiz Pedro / Resumo: No estado de São Paulo é comum aplicação de pesticidas na agricultura sem equipamentos de proteção, com grande exposição dos trabalhadores aos agentes tóxicos. O uso doméstico de inseticidas também é muito difundido. Foram demonstrados defeitos da transmissão neuromuscular, com miastenia, relacionados a pesticidas organofosforados. Inseticidas do grupo químico das piretrinas e piretróides, de uso doméstico, atuam sobre o sistema nervoso dos insetos e dos seres humanos. O objetivo deste trabalho foi procurar correlações entre Miastenia Grave e exposição crônica a pesticidas em nosso meio. Foi aplicado questionário sobre exposição a pesticidas em 217 pacientes com diagnóstico clínico e ENMG de Miastenia Grave, sendo 163 mulheres e 54 homens, com idades de 14 a 84 anos, provenientes dos serviços médicos da UNESP e UNIFESP, e em 227 controles, sendo 179 mulheres e 48 homens, com idades de 17 a 78 anos. Os dados foram analisados estatisticamente pelo Teste de Associação do Qui-quadrado. A análise estatística do total de pacientes expostos e não expostos sugeriu possível associação de exposição a pesticidas com miastenia grave (p < 0.0001) / Abstract: In the São Paulo state is very common application of pesticides in agriculture without protective equipment, with great exposure of workers to the toxic agents, and domestic use of insecticides is also widespread. Neuromuscular transmission defects with myasthenia related to the organophosphate pesticides were demonstrated in human and animals. The chemical group of pyrethrins and pyrethroids has action on the peripheral nervous system of insects and humans. The aim of this study was to search correlations between Myasthenia Gravis and chronic exposure to pesticides in our geographic region. Questionnaires were applied on exposure to pesticides in 217 patients with clinical and EMG diagnosis of Myasthenia Gravis, 163 women and 54 men, aged from 14-84 years, seen in two medical services of UNESP and UNIFESP, and in 227 controls, 179 women and 48 men, aged from 17-78 years. Data were statistically analyzed by the Association Chi-square test. The statistical analysis of all patients exposed or not exposed suggested possible association of myasthenia gravis with chronic exposure to pesticides (p <0.0001) / Mestre

Made available in DSpace on 2015-08-20T17:09:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-27. Added 1 bitstream(s) on 2015-08-20T17:26:14Z : No. of bitstreams: 1 000839732.pdf: 2196761 bytes, checksum: 01e9f7144c74e299f1fc89396be7049a (MD5) / No estado de São Paulo é comum aplicação de pesticidas na agricultura sem equipamentos de proteção, com grande exposição dos trabalhadores aos agentes tóxicos. O uso doméstico de inseticidas também é muito difundido. Foram demonstrados defeitos da transmissão neuromuscular, com miastenia, relacionados a pesticidas organofosforados. Inseticidas do grupo químico das piretrinas e piretróides, de uso doméstico, atuam sobre o sistema nervoso dos insetos e dos seres humanos. O objetivo deste trabalho foi procurar correlações entre Miastenia Grave e exposição crônica a pesticidas em nosso meio. Foi aplicado questionário sobre exposição a pesticidas em 217 pacientes com diagnóstico clínico e ENMG de Miastenia Grave, sendo 163 mulheres e 54 homens, com idades de 14 a 84 anos, provenientes dos serviços médicos da UNESP e UNIFESP, e em 227 controles, sendo 179 mulheres e 48 homens, com idades de 17 a 78 anos. Os dados foram analisados estatisticamente pelo Teste de Associação do Qui-quadrado. A análise estatística do total de pacientes expostos e não expostos sugeriu possível associação de exposição a pesticidas com miastenia grave (p < 0.0001) / In the São Paulo state is very common application of pesticides in agriculture without protective equipment, with great exposure of workers to the toxic agents, and domestic use of insecticides is also widespread. Neuromuscular transmission defects with myasthenia related to the organophosphate pesticides were demonstrated in human and animals. The chemical group of pyrethrins and pyrethroids has action on the peripheral nervous system of insects and humans. The aim of this study was to search correlations between Myasthenia Gravis and chronic exposure to pesticides in our geographic region. Questionnaires were applied on exposure to pesticides in 217 patients with clinical and EMG diagnosis of Myasthenia Gravis, 163 women and 54 men, aged from 14-84 years, seen in two medical services of UNESP and UNIFESP, and in 227 controls, 179 women and 48 men, aged from 17-78 years. Data were statistically analyzed by the Association Chi-square test. The statistical analysis of all patients exposed or not exposed suggested possible association of myasthenia gravis with chronic exposure to pesticides (p <0.0001)

This study examined the effect of neurotoxic chemical exposures on brain processes using Single Photon Emission Computed Tomography (SPECT). A control group carefully screened for good health and minimal chemical exposures was compared to two groups of patients diagnosed with health problems following exposure to pesticides or to organic solvents.

Effects of developmental exposure to chlorpyrifos (CPS) or methyl parathion (MPS) on visuospatial, adaptive fear response, and passive avoidance memory and the signaling mechanisms responsible for these neurocognitive changes were investigated. Using an incremental low dose regimen, rat pups were orally gavaged daily with either corn oil (vehicle), CPS, or MPS from postnatal day 1 (PND1) -PND21. Cholinesterase activity was significantly inhibited with the highest dosages of CPS and MPS for up to 19 days after the last dosages. OP exposure impaired working and reference memory in males whereas in the females, enhancement occurred following CPS exposure. In addition, the adaptive fear response and passive avoidance retention memory was impaired in males whereas differential changes occurred in females. Accordingly, the behavioral deficits observed in males were persistent whereas the enhancement in females was transient. Males were more sensitive to OPs than females in that the medium and high dosages of CPS and MPS produced greater effects in females whereas all dosages of both compounds produced effects in males. Training in the radial arm maze significantly increased protein kinase C gamma (PKC ) expression and activity in the hippocampal membrane fraction of control rats whereas exposure to OPs exhibited a significant decrease in PKC and PKC immunoreactivity in both untrained and trained rats. However, MPS exposed females exhibited a significant increase in PKC expression in the cytosolic fraction but this was not related to improved memory. Reduction of membrane PKC expression and activity and cytosolic PKC expression and activity seemed to be related to visuospatial learning and memory deficits in exposed males but not exposed females. Brain-derived neurotrophic factor (BDNF) gene expression in the hippocampus was significantly increased (60%) in trained control males as compared to untrained control males. In contrast, trained and untrained females exhibited similar levels of BDNF gene expression. However, exposure of both sexes to either CPS or MPS significantly reduced the expression of BDNF in trained rats. In summary, these data indicate that OP exposure induced gender-specific changes in working memory formation and altered PKC isozyme levels/activity and BDNF expression.