Alcohol consumption during pregnancy can lead to Fetal Alcohol Spectrum Disorder, and because maternal self-reports are often unreliable, a biomarker of alcohol use during pregnancy is needed to accurately determine fetal exposure. Ethyl glucuronide (EtG) is a direct metabolite of ethanol that has been detected in the meconium of infants born to mothers who consumed alcohol during pregnancy. In the current study, a method was developed and validated for EtG detection in placental perfusate and tissue using gas chromatography-mass spectrometry. Subsequently, the ex vivo human placental perfusion model was used to investigate whether EtG crosses the human placenta. The validated GC-MS method showed sufficient sensitivity in detecting EtG in placental perfusate and tissue. EtG crossed the placenta slowly and transfer was incomplete after 3 hours of perfusion. EtG appears to cross the human placenta and, hence, to represent both maternal and fetal exposure to alcohol.

Alcohol consumption during pregnancy can lead to Fetal Alcohol Spectrum Disorder, and because maternal self-reports are often unreliable, a biomarker of alcohol use during pregnancy is needed to accurately determine fetal exposure. Ethyl glucuronide (EtG) is a direct metabolite of ethanol that has been detected in the meconium of infants born to mothers who consumed alcohol during pregnancy. In the current study, a method was developed and validated for EtG detection in placental perfusate and tissue using gas chromatography-mass spectrometry. Subsequently, the ex vivo human placental perfusion model was used to investigate whether EtG crosses the human placenta. The validated GC-MS method showed sufficient sensitivity in detecting EtG in placental perfusate and tissue. EtG crossed the placenta slowly and transfer was incomplete after 3 hours of perfusion. EtG appears to cross the human placenta and, hence, to represent both maternal and fetal exposure to alcohol.

Disputats.

Alcohol has been consumed over many centuries, but its connection to criminal activity and accidental fatalities has become a prominent concern in more recent centuries(1). Scientists have developed numerous testing methods to detect alcohol consumption. Numerous studies have recently suggested that zinc has the potential to interfere with the results of these testing methods for drugs of abuse such as enzyme-linked immunosorbent assay (ELISA) and enzyme multiplied immunoassay technique (EMIT) (2, 3). False negatives have been reported from urine testing of drugs such as cocaine, methamphetamine, opiates, and cannabinoids. Nevertheless, minimal research has been conducted concerning zinc’s effect on the adulteration of alcohol metabolite testing. Ethyl glucuronide (EtG) is a promising ethanol metabolite for the confirmation of alcohol consumption. Previous research conducted by Shanna Cawley, a graduate from the Boston University School of Medicine’s Biomedical Forensic Sciences program, has found that zinc sulfate is ineffective at producing conclusive false negative results using two immunochromatographic assay brands in synthetic urine solutions(4). This study uses five different immunoassay brands, five different zinc sources, and two distinct matrices to determine the effectiveness of commercial zinc products as adulterants in drugs of abuse testing. Zinc and EtG solutions were produced at concentrations of 15mg/mL and 750ng/mL, respectively. A positive control, negative control, and two to three experimental trials were conducted for each immunoassay brand and each zinc source resulting in a total of 165 tests. Approximately sixty experimental trials in synthetic urine were invalidated or positive for the presence of EtG (81%) in zinc adulterated EtG solutions. Immunoassay kits produced false positive results when testing human urine from subjects who abstained from alcohol consumption Therefore, preliminary immunoassay screenings for the presence of EtG are not a reliable method for confirming alcohol consumption. Previously researched methods, ELISA and EMIT, and confirmatory methods such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are currently the most robust and reliable techniques for EtG detection in urine.

Doctor of Philosophy / Department of Chemistry / Ryan Rafferty / 6-Thiopurine (6TP), or 6-mercaptopurine, is an actively prescribed drug in the treatment of acute lymphocytic leukemia since 1952. Although 6TP has beneficial and promising therapeutic uses, severe toxicities are associated with its use such as jaundice and hepatotoxicity. These toxicities are due to the higher level of accumulation of bilirubin within the body. The bilirubin pathway involves the conjugation of water-insoluble bilirubin to two equivalents of UDPglucuronic acid (UDPGA) forming the water-soluble and excretable bilirubin diglucuronide species. The glucuronidation of bilirubin is catalyzed by the UDP-glucuronosyl transferase (UGT) enzyme, and the formation of substrate UDPGA is catalyzed by the UDP-glucose dehydrogenase enzyme (UDPGDH).The therapeutic activity of 6TP comes from two main routes: methylation of the thiol of 6TP and formation of a deoxy-6-thioguanosine triphosphate mimic that is incorporated into DNA resulting in apoptosis. In conjugation to its therapeutic metabolism, there are also detoxification pathways operating simultaneously that significantly reduce its therapeutic activity. In this pathway, oxidative metabolites of 6TP such as 8-hydroxyl-6-thiopurine (6TP-8OH), 6-thioxanthine (6TX) and 6-thiouric acid (6TU) can be formed by xanthine oxidase. It has been observed that the body retains 6TU well beyond 24-hour post 6TP treatment. Therefore, we proposed that the observed toxicity from 6TP administration comes from either 6TP or its oxidative excretion metabolites’ ability to inhibit one or both enzymes (UDPGDH, UGT) in the bilirubin excretion pathway. To investigate the toxicity resulting from the 6TP administration about these two enzymatic steps, inhibition analysis of these oxidative metabolites on UDPGDH was assessed using a robust UV-Vis method. The inhibition profile made with regards to varying UDP-glucose showed weak to no inhibition of 6TP towards UDPGDH with a K[subscript]i of 288 μΜ. However, 6TU, (the primary oxidative metabolite which is oxidized at C2 and C8) has increased inhibition towards UDPGDH with K[subscript]i of 7 μΜ. Inhibition was also observed with 6TX (oxidized at C2) and 8-OH-6TP (oxidized at C8) with K[subscript]i values 54 and 14 μΜ, respectively. To further confirm the results of the UV-Vis assessment, inhibition studies were carried out using an HPLC method that was developed and validated to separate all the analytes in the UDPGDH catalyzed reaction. Inhibition studies were performed via the HPLC method showed K[subscript]i values of 105 μΜ and 5 μΜ for 6TP and 6TU, respectively, towards UDPGDH. To assess the inhibition studies towards the UGT enzyme, an HPLC method was developed for the simultaneous determination of bilirubin and its mono/diglucuronides. The inhibition studies were carried to assess the formation of glucuronides and consumption of UDPGA in the presence of the inhibitors using the HPLC method developed. Neither 6TP nor 6TU were shown to inhibit UGT. Also, inhibition studies were carried out in vivo animal model, which further confirmed that 6TP and 6TU do inhibit UDPGDH, but no effect on UGT activity. With these results, we discovered that both 6TP and its oxidative metabolites inhibit UDPGDH. Furthermore, it was observed that C2 and C8 positions of 6TP are important for the toxicity towards UDPGDH. With the goal of developing a single multi-enzymatic assay, another HPLC method was developed to assess the UDPGDH and UGT catalyze reactions together. This method can be used as a standard method to assess interference of any molecule on bilirubin excretion. Given the findings in this study, efforts are being directed towards the synthesis of 8-substituted 6TP analogs that are proposed to retain its therapeutic efficacy but have limited to no toxicity.

Morphine has been and continues to be one of the most potent and widely used drugs for the treatment of pain. Clinical and animal models investigating sex differences in pain and analgesia demonstrate that morphine is a more potent analgesic in males than in females; indeed, we report the effective dose of morphine for female rats is twice that of male rats. In addition to binding to the neuronal mu opioid receptor, morphine binds to the innate immune receptor toll-like receptor 4 (TLR4) on microglia. Morphine action at TLR4 initiates a neuroinflammatory response and directly opposes morphine analgesia. Our recent studies demonstrate that administration of chronic morphine activates microglia within the ventrolateral periaqueductal gray (vlPAG), a critical brain region for the antinociceptive effects of morphine, while blockade of vlPAG microglia increases morphine analgesia and suppresses the development of tolerance in male rats. Despite increasing evidence of the involvement of microglia in altering morphine efficacy, no studies have examined sex differences in microglia within the PAG. The present experiments seek to characterize the distribution and activity of vlPAG microglia in males and females using behavioral, immunohistochemical and molecular techniques, while demonstrating the sufficiency and necessity of vlPAG microglia to produce sex differences in morphine analgesia using site-specific pharmacological manipulation of TLR4. We also investigate a novel pharmacokinetic mechanism underlying the sexually dimorphic effects of morphine administration on microglial activity. Here, we address a fundamental gap in our current understanding of sex differences in morphine analgesia and establish a mechanistic understanding of how the activation of vlPAG microglia sex-specifically influences morphine analgesia.

L’exposition au bisphénol A (BPA) au cours du développement fœtal est suspectée d’être impliquée dans l’initiation d’effets biologiques. Dans ce contexte, l’objectif de cette thèse est d’évaluer l’exposition humaine fœtale au BPA. Une étude pharmacocinétique (PK) réalisée sur le modèle du fœtus ovin a montré que le BPA glucuronide (BPAG), métabolite majeur du BPA piégé dans le compartiment fœtal, est lentement éliminé via sa réactivation en BPA. A partir de ces données PK, le développement d’un modèle PK humanisé a permis de prédire des concentrations plasmatiques fœtales de BPAG maintenues autour de 40ng/L chez le fœtus humain en fin de grossesse, soit environ 1000 fois supérieures aux concentrations en BPA correspondantes. Une analyse protéomique de tissus fœtaux ovins a montré qu’une même dose molaire de BPAG ou de BPA affecte des voies physiologiques similaires, suggérant que l’hydrolyse du BPAG pourrait exposer les tissus à des concentrations en BPA compatibles avec l’expression d’un effet biologique. L’ensemble de ces résultats indique que les concentrations de BPAG dans le sang de cordon constituent un marqueur pertinent de l’exposition fœtale au BPA et qu’elles doivent être prises en compte dans l’évaluation du risque / Prenatal exposure to bisphenol A (BPA) is suspected to induce adverse effects later in life. In this context, the goal of this thesis was to evaluate the human fetal exposure to BPA. Using a pharmacokinetic (PK) approach, we showed that BPA glucuronide (BPAG), the main metabolite of BPA that remains trapped in the fetal compartment, is slowly eliminated through its back conversion into BPA. A humanized PK model developed from these data predicted steady BPAG fetal plasma concentrations of 40ng/L in late pregnancy, i.e. about 1000 folds higher than corresponding BPA concentrations. A proteomic analysis of fetal tissues has shown that equimolar BPAG or BPA fetal exposures trigger similar physiological shifts in the ovine model, suggesting that BPAG hydrolysis might expose fetal tissues to BPA at levels of functional significance. It is concluded that BPAG levels in human cord blood are relevant indicators of fetal exposure to BPA during late pregnancy and should be taken into account for risk assessment

Morphine-6-glucuronide is a minor metabolite. representing 5% of an administered dose of morphine. The metabolite has analgesic activity exceeding that of morphine and may contribute to analgesia following morphine administration. The aims of the study were to attempt to identify the reasons behind the improved activity of morphine-6-glucuronide over the parent compound and to examine a series of 6-substituted compounds, based on 6-substituted benzoate esters, as potential mimics of morphine-6-glucuronide. Morphine-6-glucuronide was seen to have similar affinity to morphine at l1-opioid receptors as assessed by ligand-binding assays in mouse brain homogenates. However a three-fold improved affinity at S-opioid receptor binding sites was observed and a ten-fold reduction in affinity at K-opioid sites. Using in vitro bioassay systems the glucuronide showed a two-fold improved potency over morphine in both the guinea-pig ileum and the mouse vas deferens preparations. Following in vivo (s.c.) administration in the mouse the glucuronide was seen to be equipotent with morphine in the tail-flick test, but was of much longer duration, lasting up to 9 hours. Exvivo binding assays confirmed that morphine-like material was still present in the central nervous system six hours after administration of the glucuronide, but was not observed at a similar time after morphine administration. Activity was retained if the hydroxyl groups of the sugar moiety of the glucuronide were protected as esters. In contrast the more prevalent morphine metabolite morphine-3-glucuronide was inactive in all in vitro and in vivo tests used and did not antagonise morphine in vitro or in vivo. A group of 3-substituted derivatives containing saturated and unsaturated substituents did show affinity for opioid receptors but no agonist activity of the compounds could be demonstrated in vitro. A series of synthetic 6-substituted compounds showed a variety of affinities for, and agonist potencies at, opioid receptors, though low affinity at Kopioid receptors was a general finding. For example, morphine-6- nitrobenzoate was l1-opioid receptor preferring, while morphine-6- phthalate had improved O-opioid receptor affinity and acted via Il-opioid receptors in the mouse vas deferens and in vivo. However the compounds were weaker than morphine and the duration of action in vivo was shorter than morphine-6-glucuronide. The conclusions from these studies are that morphine-6-glucuronide and morphine have similar in vitro affinities at the l1-receptor, although morphine-6-glucuronide has somewhat improved binding affinity for Il receptor sites, it has less affinity for K receptor sites. Pharmacokinetic reasons are probably responsible for the improved activity and duration of action of morphine-6-glucuronide over morphine. None of the synthetic compounds examined are potentially useful as direct mimics of the glucuronide because morphine-6-glucuronide is more potent and has a longer duration of action than the synthetic derivatives, though alteration at the 6-position of the morphine nucleus can lead to dramatic changes in selectivity and potency of ligands for the differing opioid receptors.

No description available.

>Magister Scientiae - MSc / Informal settlements are often implicated in surface water pollution with faecal matter. In most instances faecal pollution in the associated surface waters persists despite improvements in sewage removal infrastructure. This study evaluates the importance of investigating the water quality of the Plankenbrug River before it reaches Khayamnandi settlement by comparing water quality in spring and in winter upstream (Pre-Khayamnandi) and downstream (Post- Khayamnandi) from the settlement. In this study, faecal indicator bacteria (Escherichia coli and total coliforms) were enumerated using Chromocult agar. E. coli was further characterized with analytical profiling index (API) and haemolysis assays. Both Pre- and Post-Khayamnandi were not significantly different from each other for both total coliforms and E. coli in winter. Pre-Khayamnandi had between 105 and 108 cfu/100 ml for total coliforms while Post-Khayamnandi had total coliform colony count between 106 and 107 cfu/100 ml. E. coli also exhibited a similar pattern with slightly higher counts at Post-Khayamnandi with colony counts from 104 to 107 and 105 to 107 cfu/100 ml. Spring microbial count demonstrated a significant difference to winter counts within each test site (p ≤ 0.01) and across the two sites (p ≤ 0.05). Both total coliforms and E. coli were 102 fold higher at Post-Khayamnandi than at Pre-Khayamnandi in spring. The API assay demonstrated significant difference (p ≤ 0.05) between the two test sites. Pre- Khayamnandi predominantly had two different profiles while Post-Khayamnandi had three. These profiles represented five distinct E. coli biotypes. Sorbitol and sucrose tests within the API assay demonstrated significant differences (p ≤ 0.05) between the two test sites. The prevalence of sorbitol fermenters at Pre-Khayamnandi was 100% while at Post-Khayamnandi it was 73%. Pre-Khayamnandi also demonstrated a significantly higher prevalence of sucrose fermenters than Post-Khayamnandi at 100% and 59% respectively. These differences indicated dissimilar sources of faecal contamination around these sites. Differences in the distributions of sorbitol and sucrose fermenting biotypes demonstrate different toxicity potentials across these two test sites. The haemolysis assay demonstrated that 9% of isolates were haemolytic with reference to both known α- and β-haemolyitic streptococci at Post-Khayamnandi. At Pre-Khayamnandi there was a higher percentage of α- and β-haemolyitic species, 29% and 28%, respectively. Post- Khayamnandi and Pre-Khayamnandi were significantly different from each other with reference to both α- and β-haemolysis (p ≤ 0.05). These haemolytic activities also demonstrate different toxicity potentials across the two sites. In conclusion Khayamnandi contributes to an already heavy faecal load in the Plankenbrug River. Thus remedial measures to maintain high surface water quality of Plankenbrug River should be directed upstream from the Khayamnandi settlement as well as within the settlement equally. This study recommends integration of microbial loads with programs such as the National Microbial Monitoring Program of South Africa to drive prioritization process in directing reclaiming of water quality, inter alia.