Xenoestrogens are chemical pollutants that can disrupt the endocrine system of animals by binding to and activating the estrogen receptor(s). They include both natural and synthetic steroid estrogens, together with a variety of estrogen mimicking chemicals such as 4-nonylphenol, bisphenol A and various pesticides. In vertebrates, estrogens play a fundamental role in reproduction, in somatic cell function, the regulation of calcium and water homeostasis. Exposure to xenoestrogens may therefore have unscheduled effects on these systems that can potentially compromise species survival. With the ever-increasing number of xenoestrogens identified and detected in the environment, together with the fact that they are seldom detected alone, there is a need to develop specific and sensitive biomarkers to detect estrogenic activity of chemicals in the environment when present alone and in mixtures. In this study, the effect of selected xenoestrogens was assessed using an in-vitro yeast estrogen screen (YES) both individually and in mixtures and in-vivo in a native fish species, the Murray rainbowfish (Melanotaenia fluviatilis).

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains viii, 69 p. : ill., maps. Includes abstract. Includes bibliographical references.

O monóxido de carbono (CO) constitui sério risco à saúde de indivíduos expostos a este gás. Os efeitos nocivos aparecem como conseqüência de sua combinação com a hemoglobina formando a carboxiemoglobina(COHb). A avaliação da exposição ao CO pela monitorização biológica é realizada, preferencialmente, pela determinação da carboxiemoglobinemia. O método espectrofotométrico proposto, para a determinação de COHb, utiliza a leitura na região Soret (420 e 432 nm) e fatores de calibração do espectrofotômetro. É realizado estudo comparativo entre o uso do CO obtido por reação química e o de cilindro de gás no preparo de solução 100% de COHb. O método apresenta boa precisão (coeficiente de variação de 2 e 6% para 4,98 e 1,01 % de COHb, respectivamente) e sensibilidade (0,50 % de COHb) adequadas à avaliação da exposição ao monóxido de carbono. A quantificação da COHb não é comprometida pelo teor hemoglobínico nem pela opalescência (lipemia) da amostra colhida no período pós-prandial. É apresentada a carboxiemoglobinemia em fumantes (n= 119) e não-fumantes (n= 189) que constituem 4 grupos de indivíduos expostos ocupacionalmente ao CO (n=209) e um grupo controle (n=99); e a análise estatística dos resultados (teste não-paramétrico). / Carbon monoxide (CO) is recognized as a high risk hazard to the health of exposed workers. Combining with hemoglobin it reduces the oxigen carrying capacity of the blood. The individual overall exposure may be assessed through the carboxyhemoglobin (COHb) content of blood samples, as a biological exposure index. A spectrophotometric method is proposed using measurements in the region Soret (420 - 432 nrn) together with calibration factors of the instrument. A comparative study is made between the use of CO from compressed gas cilinders and the CO delivered by a chemical reaction in preparing the saturated COHb solution. The method presents precision (coefficient of variation is 2 and 6% to 4,98 and 1,01% of COHb, respectively) and sensitivity (0,5% of COHb), which are adequate to the purpose. Hemoglobin and lipidic content of samples showed no effect in the COHb measurement. Carboxyhiemoglobin level of four groups of exposed workers (n = 209) and a control group (n = 99) among smokers (n = 115) and non-smokers (n = 189) were determinated using the method. The statistical analysis of the results are presented (non-parametric test).

D.Sc. (Zoology) / The influence of some natural enemies and pesticides was studied on Tetranychus cinnabarinus (Boisduval), T. lombardinii Baker & Pritchard and T. ludeni Zacher (Acari: Tetranychidae). Two important predators were discovered, viz: Pardosa crassipalpis (Purcell) (Arachnida: Araneida) and Labidura riparia (Insecta: Dermaptera). Both are more susceptible to some acaricides, especially dimethoate, than the mites. It is proved that the injudicious use of these acaricides, also of DDT and carbaryl, can cause an outbreak of these mites. As to the biologies of the species, the appearance was noted of a fourth chrysalis stage during the development of three T. cinnabarinus females.

D.Phil. (Chemistry) / Please refer to full text to view abstract

Liquid phase catalytic hydrodechlorinations can provide a convenient and environmentally friendly method for treating organic chlorinated compounds in waste streams generated during the manufacturing of agrochemicals. During such treatment hydrochloric acid is generated as a by-product, which can be easily neutralized employing a base to yield an inorganic salt. This work describes the results obtained during the liquid phase hydrodechlorination of 2,6-dichlorophenol (2,6-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP). The hydrodechlorination of these chlorinated phenolic compounds in a mixture of methanol and water was investigated using supported and unsupported palladium catalysts to yield lower chlorinated phenols or phenol. Various parameters were investigated such as catalyst concentration, ammonium formate concentration, effect of base addition and effect of temperature. During this study two methods of hydrodechlorination were also investigated such as hydride-transfer hydrogenolysis, using ammonium formate, and hydrogenolysis, using dihydrogen. These methods offer a mild treatment in terms of the reaction temperature with temperatures used below 800C. A comparison of the palladium catalyst systems using these methods also showed that Pd/C produced the best results in terms of the hydrodechlorination rate and the selectivity towards phenol. When the ammonium formate method was used, complete hydrodechlorination of both substrates was achieved in 1h of reaction time at a selectivity of 100 percent towards phenol. A comparison of the method using dihydrogen and Pd/C showed that the reaction rate and the selectivity towards phenol during the hydrodechlorination of 2,6-DCP were 87.92 percent and 93.30 percent. Similarly, the reaction rate and selectivity during 2,4,6- TCP hydrodechlorination were 63.77 percent and 70.57 percent. These results were achieved in a reaction time of 3 hours. A high catalyst loading increases the reaction rate at the expense of selectivity, due to the formation of cyclohexanone, formed during further hydrogenation of phenol. The formation of cyclohexanone was limited at high temperatures (ca. 800C) with none detected during the hydrodechlorination of 2,6-DCP and 0.19 percent during the hydrodechlorination of 2,4,6-TCP. Evaluation of the hydrodechlorination parameters showed that the catalytic efficiency of the Pd/C catalysts was inhibited as the reaction proceeded due to the formation of HCl as by-product. A significant increase in the reaction rate was achieved when the reaction was performed in the presence of an inorganic base, which neutralized HCl.

The three isomers of dichlorobenzene (DCB) exhibit marked differences in hepatotoxicity following intraperitoneal (ip) administration in male F-344 rats. Plasma GPT activity, measured 24 hours post exposure, was elevated to approximately 4080 units/ml following a 1.8 mmol/kg dose of o-DCB. Conversely, n-DCB produced only a moderate elevation (306 units/ml) following a 4.5 mmol/kg dose, while p-DCB produced no elevation in GPT activity at this dose (24 units/ml). Ultra-structurally, o- and m-DCB induced elevations in GPT activity were associated with a centrilobular pattern of hepatic necrosis. The role of cytochrome P-450 mediated bioactivation in DCB-induced hepatotoxicity was demonstrated by elevated GPT activities following an otherwise nontoxic 0.9 mmol/kg dose of either o- or m-DCB in phenobarbital pretreated animals (16770 and 21540 units/ml, respectively). The paraisomer of DCB showed no induction of toxicity with phenobarbital pretreatment. Hepatic glutathione (GSH) concentrations were reduced 0.5, 3 and 5 hours after a 1.8 mmol/kg dose of either o- or m-DCB, a dose which produces hepatotoxicity only for o-DCB. Pretreatment of animals with phorone depleted hepatic GSH to 15% of control levels within two hours; subsequent ip administration of either o- or m-DCB (1.8 mmol/kg) produced approximately equivalent elevations in GPT activity for both isomers (5749 ± 648 and 4732 ± 857 units/ml, respectively). In vitro incubations of o- and m-DCB with GSH and rat liver cytosolic fraction, suggested that GSH may bind m-DCB without prior bioactivation, thereby reducing the hepatotoxicity of this isomer relative to the more toxic ortho isomer. The interactive hepatotoxicity of the dichlorobenzenes with carbon tetrachloride (CCl₄) was also investigated. Concomitant ip injection of CCl₄ (1.0 mmol/kg) and o-DCB (2.7 mmol/kg) produced a marked inhibition of o-DCB hepatotoxicity, as measured by GPT activity (approximately 200 units/ml vs. 7450 units/ml for o-DCB alone). The mechanism of this inhibition of o-DCB hepatotoxicity was shown to be a reduction in the cytochrome P-450 mediated bioactivation of o-DCB, by CCl₄. A similar inhibition of o-DCB hepatotoxicity was seen following administration of CCl₄ as a pretreatment, via the drinking water. Concomitant ip administration of CCl₄ with either m- or p-DCB also produced a reduction in the metabolism of the dichlorobenzene.

Forensic toxicology is a critical field in which scientific techniques are employed in order to establish the presence or absence of pharmacological substances and/or their metabolites within an individual. The results of such analyses can have legal implications, and toxicology has a number of important applications, including post-mortem investigations, workplace drug testing, therapeutic drug monitoring, and impaired driving studies. The focus of this specific body of work is on the use of toxicology in the detection and quantification of drugs of abuse –specifically opioids - in biological samples. In recent years, there has been a surge in opioid abuse and the need for forensic toxicology labs to process samples from such cases quickly and accurately continues to increase. As a result, it is imperative to research different techniques and technologies that can be applied in toxicology to improve efficiency of sample processing while still remaining sensitive and specific. Many toxicology laboratories today use immunoassay techniques for screening, and utilize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification. While these methods are established and reliable, the need to analyze an increasing number of samples in a more efficient time frame is essential, and with that, the need to develop and validate new analytical methods. This study sought to validate the use of Microchip Capillary Electrophoresis-Tandem Mass Spectrometry (CE-MS/MS) as a method for detecting and quantifying a panel of fourteen opioids. The experiments were run using a ZipChip (908 Devices, Boston, MA) as the separation scheme, which contains a small capillary where analytes are separated out by electrophoretic mobility - dictated largely by size and charge. These analytes were then ionized by electron spray ionization (ESI) at the end of the chip, and then detected, fragmented, and analyzed in a SCIEX 4500 Triple Quadrupole Mass Spectrometer (Framingham, MA). The analytical run time of the method evaluated was two and half minutes per sample. Calibration curves were run and the method was assessed for a number of validation parameters, including bias, precision, limit of detection, common analyte interferences, matrix interferences, and carryover, as recommended by the American Academy of Forensic Sciences Standards Board. The fourteen drugs and metabolites looked at in this study were 6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine (EDDP), fentanyl, heroin, methadone, morphine, naloxone, norfentanyl, oxycodone, oxymorphone, and tramadol. All standards were ordered from Cerilliant (Round Rock, TX), as well as deuterated internal standards used for quantification purposes. This study showed that as the method currently stands, it can reliably detect this panel of opioids at limits of detection between 1 and 15 ng/mL, with the exception of buprenorphine and morphine, for which the method appeared less sensitive. While some applications desire higher sensitivity than this, this level of detection could be very useful as a screening technique that is quick and also far more specific than current immunoassay screening techniques, and provide the additional advantage of quantification for samples at slightly higher concentrations. Quality control samples at 100 ng/mL and 150 ng/mL generally showed consistent results and acceptable levels of bias and precision, indicating that the method can be used to reliably quantify this panel of opioids at those concentrations. In addition, interference signals detected during analysis of other common analytes often encountered with opioids were negligible, with the exception of heroin and norfentanyl. Analysis of ten lots of urine for blank matrix interferences also demonstrated low potential for interference, with the exception of heroin. Finally, there was no evidence of significant carryover between samples, or interference from the deuterated internal standards. While some potential instrumentation issues such as mass spectrometer calibration prompt further study, the method shows promise for future use as a high throughput analysis tool in forensic toxicology labs. CE-MS/MS has the added benefit of not only faster run times, but significantly less sample consumption per run, and additionally, less sample preparation. CE is a viable separation scheme for metabolites and forensic applications, and could make large impacts as an effective way to analyze toxicological samples.

<p>   </p> <p>Copper (Cu) dyshomeostasis in the brain, especially Cu overload, has been associated with neurodegenerative disorders such as Alzheimer’s disease (AD). Brain Cu levels are partly regulated by the choroid plexus (CP), a tissue that forms a barrier between the blood and cerebrospinal fluid (CSF) and is rich in Cu-transporting proteins. Literature data have shown that ADP-ribosylation factor 1 (ARF1) plays a role in regulating cellular Cu homeostasis; yet its presence and function in the blood-CSF barrier was unknown. The main purpose of the project was to prove the presence of ARF1 in the CP and explore its possible function with regards to Cu regulation. Since the CP is known to accumulate toxic metal lead (Pb) from human and animal studies, the project also aimed to test whether Pb exposure caused CSF Cu dyshomeostasis through disrupting ARF1-mediated Cu regulatory mechanism. Using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), our data clearly demonstrated that ARF1 was highly enriched in the choroidal epithelial Z310 cells and expressed in CP tissues. Acute Pb exposure in mice (one ip. dose at 27 mg Pb/kg followed by tissue dissection 24 hrs later) significantly increased ARF1 expression in the CP as compared to saline-injected controls, suggesting a high responsiveness of the protein to Pb exposure. In the subsequent chronic study, mice were exposed to Pb via drinking water at 200ppm (low-dose) or 800ppm (high-dose) at libitum for 4 weeks (control group had sodium in drinking water). Atomic absorption spectrometry (AAS) analyses verified a dose-dependent Pb accumulation in the CP. Importantly, Cu concentrations in the CSF displayed a Pb dose-dependent increase as compared to controls. IHC data further revealed an altered ARF1 expression in the choroidal epithelia in chronic Pb exposed animals. Further knocking down ARF1 expression in choroidal epithelial Z310 cells using in vitro Arf1-targeting siRNA transfection approach revealed a decreased Cu level in cells, suggesting a critical role of ARF1 in cellular Cu regulation. In choroidal epithelial cells exposed to Pb in culture medium, a down-regulated ARF1 expression partly reversed Pb-induced Cu overload. Taken together, this study provides first-hand evidence to support the presence of ARF1 in the blood-CSF barrier. Further, our data demonstrate that Pb exposure causes Cu overload in the CSF, which is likely mediated by an altered expression of ARF1 in the blood-CSF barrier.</p>

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