| Abstract |
The purpose of the present PhD dissertation was the in vivo assessment of long term exposure to sub-acute doses of organophosphate pesticides Diazinon {O,O-diethyl-O-(2-isopropyl-4methyl-6-pyrimidinyl)} and Chlorpyrifos-ethyl {O,O-diethyl-O-3,5,6-trichloro-2-pyridylphosphorothioate} in terms of determination of the levels of their metabolites in hair. The influence of dose and exposure duration of each pesticide on the concentrations of the non specific metabolites DEP and DETP in hair was also investigated. Also, their role as appropriate biomarkers for the assessment of chronic and past exposure was evaluated. Determination of Diazinon, Chlorpyrifos-ethyl and Propoxur residues in rabbit heart tissue was conducted in order to investigate their cariotoxic effect. In addition, all pesticides were tested to evaluate their potential of inducing oxidative stress and oxidative modifications in the genomic DNA content of the cardiac tissues. The present PhD thesis is constisted of five sections.
The first section consists of the in vivo study protocol of exposure to organophosphates Diazinon and Chlorpyrifos-ethyl in rabbits (N=12). Rabbits were divided into four treatment groups of three animals each and were treated with two sub-acute doses of diazinon (3.0, 6.0 mg /kg /day) for four months and with a single dose of Chlorpyrifos-ethyl (18.0 mg /kg /day) for three months. Sample collection was performed monthly during the treatment period. Hair samples were also collected seven months after the last dose for diazinon treated animals.
The second section describes the procedure of collection and processing of hair samples. During in vivo experimental project, samples of rabbit hair were collected for the measurement of the pesticides metabolites levels and the assessment of chronic accumulative exposure. Before the beginning of the dosage period, specific parts of the animals back were shaved. Sampling began a month after the first dose administration and samples were collected at the end of every month of the dosage period and seven months after the last in the case of Diazinon treated test animals. Also, urine, blood and faeces samples were abtained at the end of every month.
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In the next part, the conditions of detection and quantitive analysis of the non specific dialkylphosphates metabolites of the organophosphate Diazinon and Chlorpyrifos-ethyl, in the hair shaft, are described. Specifically, the levels of diethylthiophosphate (DETP) and diethylphosphate (DEP) were estimated at the end of every month of the exposure period by the analytical procedures which included decontamination step, solid-liquid extraction, derivatization process and gas chromatography tandem mass spectrometry (GC-MS) analysis. During the extraction procedure the parameters of extraction duration, temperature and sonication duration were evaluated. The suitability of the exraction solvent was also investigated in regard to the polarity of the analytes DEP and DETP. For GC-MS analysis, a previously proposed method was employed. The procedure was shown to be linear over a wide range of concentrations. The obtained recoveries in rabbit hair were 85 % and 105% for DEP and DETP, respectively. Quantification limits were 15 pg / mg and 37 pg / mg for DEP and DETP respectively.
In the third section, the results of analysis of the hair samples are discussed in detail. The mean levels of DEP and DETP in the hair samples of Diazinon treated animals range from 112 to 585 pg / mg and 295 to 988 pg / mg for low and high level dose respectively throughout the four months of exposure. The mean levels of DEP and DETP, obtained from the Cholorpyrifos-ethyl treated animals, ranged from 138 to 1070 pg / mg and 554 to 886 pg / mg respectively during the three months of exposure. Also, the analysis of hair samples seven months after the last Diazinon administration showed that the mean levels of DEP and DETP are within the range of values recorded for the 1-month-exposed sample.
Results of hair analysis revealed correlation between Diazinon dosage and detected concentrations of the non specific metabolites DEP and DETP. This seemed to be significant, certainly for DEP, while a tendency is observed for DETP. The quantification of diethyl phosphates also, manifests that increase of the duration of exposure to Chlorpyrifos-ethyl and Diazinon is associated with an increase of accumulation of DEP and DETP in hair samples. These data confirms the ability of hair analysis to assess the chronic exposure to low levels of organophosphates and the usefulness of the employed method of analysis for such purpose.The detected values of
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diethyl phosphates several months after the last dose administration suggest that hair could be a valuable tool for the assessment of past exposure to pesticides.
In the last part, the aim of the study was to assess the effects of long term and low-level exposure to these pesticides on the heart function, simulating the occupational exposure of the agricultural workers. Another eight rabbits were divided into four groups consisting of two animals each. The groups of rabbits were exposed to two different doses of Diazinon (2.6-5.2 mg/Kg /day), Chlorpyrifos-ethyl (8.7-18 mg/Kg/day) and Propoxur (8.8-17.5 mg/Kg/day). Several echocardiographic techniques were used for the assessment of myocardial size and performance. The main findings suggested the presence of impaired systolic and diastolic myocardial function, impaired cardiac index as well as reduced myocardial mass in the pesticide-treated animals compared to controls. Also, histopathologic analysis of cardiac muscle tissue was performed in order to identify possible alterations. The main findings were fibrosis and hemorrhagic infiltration. The levels of localized and systemic oxidative stress parameters like TBARS, protein carbonyls, catalase activity and TAC were also investigated. The data suggest that all tested pesticides induced oxidative stress. At last, all the pesticides were tested for inducing oxidative DNA modifications and the results revealed that they caused significant DNA modifications.
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Δείκτες χρόνιας έκθεσης στους οργανοφωσφορικούς εστέρες.Μελέτη εναπόθεσης μη ειδικών μεταβολιτών σε δείγματα τριχών πειραματόζωων .Ποιοτική και ποσοτική ανάλυση με αέρια χρωματογραφία/φασματομετρία μάζας(GC-MS)
