Mycobacterium avium causes disseminated infection in humans with immunodeficiency, pulmonary infections in individuals with predisposing lung conditions (e.g., pneumoconiosis), and cervical lymphadenitis in children. Twenty-five to fifty percent of late stage AIDS patients are infected with M. avium. M. avium has been recovered from drinking water and strains from water share identical DNA fingerprints with isolates recovered from patients exposed to the water. Further, M. avium is resistant to chlorine, a disinfectant commonly used in municipal water supplies. Because of the slow growth of M. avium, measuring its susceptibility to disinfectants is laborious and reaction to a potential problem is delayed. Thus, there exists a need for a rapid test to measure the antimycobacterial disinfectant capability of chlorine containing water samples. The objective of this research was to develop a rapid and quantitative assay for the viability of mycobacteria using firefly luciferase as a reporter gene for disinfection survival studies. Derivatives of M. avium strains MD1 and A5, Mycobacterium smegmatis strain VT307 and Mycobacterium bovis BCG strain Pasteur carrying the firefly luciferase gene (pLUC10) were constructed. In pLUC10-carrying strains of M. avium strain A5 and M. smegmatis strain VT307, a direct correlation was shown between the quantity of light produced and the number of cells recovered as colony forming units. In disinfection studies of both pLUC10-carrying derivatives of M. avium strain A5 and M. smegmatis strain VT307, survival, as measured in colony forming units, correlated with survival in relative light units. Luciferase measurements appear to offer a method for rapid enumeration of mycobactericidal disinfection capacity of chlorinated water. / Master of Science

PXR mediated elevation of CYP3A4 expression is a costly problem in drug development as well as a clinical problem due to clinically important drug interactions caused by the enzyme induction. CYP3A4 is responsible for the metabolism of more than 50% of the drugs commonly used today. Many of these, as well as other compounds e.g. in herbal medicines can induce transcription of CYP3A4 and thereby enhance the metabolism of other drugs, rendering them ineffective or more toxic. By using an in vitro assay for CYP3A4 induction, tests can be performed on candidate drugs early in development and thereby save time and resources since CYP3A4 inducers are eliminated from further development. A reporter gene assay was constructed by inserting three modules, which includes PXR binding sites isolated from the CYP3A4 sequence, in front of a luciferase gene. This construct was transfected together with PXR into HEK 293 cells. Induction was evoked by adding rifampicin, a known CYP3A4 inducer, to the medium. After lysis of the HEK cells and addition of luciferase substrate, luminescence intensity was recorded as a measure of induction. The construct worked and consistently showed induction by rifampicin, but could be further improved to yield higher sensitivity.

<p>PXR mediated elevation of CYP3A4 expression is a costly problem in drug development as well as a clinical problem due to clinically important drug interactions caused by the enzyme induction. CYP3A4 is responsible for the metabolism of more than 50% of the drugs commonly used today. Many of these, as well as other compounds e.g. in herbal medicines can induce transcription of CYP3A4 and thereby enhance the metabolism of other drugs, rendering them ineffective or more toxic. By using an in vitro assay for CYP3A4 induction, tests can be performed on candidate drugs early in development and thereby save time and resources since CYP3A4 inducers are eliminated from further development. A reporter gene assay was constructed by inserting three modules, which includes PXR binding sites isolated from the CYP3A4 sequence, in front of a luciferase gene. This construct was transfected together with PXR into HEK 293 cells. Induction was evoked by adding rifampicin, a known CYP3A4 inducer, to the medium. After lysis of the HEK cells and addition of luciferase substrate, luminescence intensity was recorded as a measure of induction. The construct worked and consistently showed induction by rifampicin, but could be further improved to yield higher sensitivity.</p>

No description available.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

The project involved the study of 12 Victorian municipal wastewater treatment plant discharges. These included lagoon-based plants and those with activated sludge based processes. Permission was obtained from all the relevant water authorities to collect samples of final effluent at point of discharge to the environment, whether that was to a creek, a river, the ocean, or the land. Samples were collected in November 2003, and then again in April and June 2004, and subjected to a number of biological and chemical analyses, including toxicity tests, measurement of hormonal (estrogenic) activity using yeast-based bioassays, and the measurement of specific hormonal concentrations (17-estradiol) using enzyme-linked immunosorbent assays (ELISA). Almost all of the effluents examined showed estrogenic activity, to a greater or lesser extent (no response to 55 ng/L 17β-estradiol equivalents). On the whole, the levels of estrogenic activity observed were to the lower end of the range observed overseas in the northern hemisphere, and comparable with that recently reported in Australia and New Zealand using similar, human-estrogen receptor based assays (no response to ~ 10 ng/L 17β-estradiol equivalents). The reassuring low/no assay response is bolstered by the chemical assessment of estradiol concentrations by ELISA, which returned concentrations of these compounds for the most part in the range 2-5 ng/L. From an aquatic environmental perspective, it is difficult to say with any certainty what the potential risk to aquatic organisms in waters receiving these effluents will be. Typically, in environmental risk assessment one first looks to agreed national or international guideline or trigger values for the type of waters being assessed. In this case, there are as yet no guideline values. Without guideline values to drive the assessment, then one compares a chemical’s concentration in a sample (in this case a WWTP effluent) with data obtained from toxicological experiments in which the concentration known to elicit a specific effect has been determined. In this case, levels of 17β-estradiol were typically between the lowest reported level to induce the production of Female-indicative proteins in male fish (plasma vitellogen; 1 ng/L), and the lowest concentration of known to induce intersex in fish (8 ng/L). Consequently, such levels in a WWTP discharge are likely to be an environmental risk if there is little or no dilution of the discharge by the receiving water, i.e. discharge represents major component of stream flow. In short, to truly assess the risk (hormonal impact) of these WWTP effluents, in vivo testing needs to be undertaken, ideally with a representative native species but failing that with a ‘standard’ species such as the fathead minnow. When this programme began, the ‘watching brief’, being held in Australia on the topic of endocrine disrupting chemicals and their potential effects on aquatic wildlife was considered too passive by many. It still is, by some. Despite the assurance the results may provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground, reassurance research to provide data for higher-level risk assessment by industry and government agencies.

POPs are a group of chemicals that have been extensively studied over the last few years. The main reason that these chemicals have received so much scientific attention is the myriad of negative effects they have on the environment and human health. The properties that cause the deleterious effects include a high molecular stability, rendering them highly persistent. Added to this is the lipophilic and hydrophobic nature of the compounds. POPs will thus tend to bio-accumulate and bio-magnify in the environment, causing a direct threat to humans and wildlife. To address this threat, the Stockholm Convention on Persistent Organic Pollutants, under the supervision of United Nations Environment programme (UNEP), was initiated and became legally binding on 17 May 2004. All countries, including South Africa, which ratified this agreement, will be expected to monitor and regulate the formation of POPs. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are all members of the dioxin-like family of POPs. This family of chemicals pose serious health threats such as carcinogenic effects and negative effects on reproduction. These substances, with the exception of PCBs, are formed unintentionally as by-products of industrial and thermal processes. One of the main sources of dioxin-like chemicals is medical waste incinerators. In this project the area surrounding a medical waste incinerator was monitored using a bio-assay technique. The determination of dioxin concentrations is usually preformed by chemical analysis, however, bio-assays have proven themselves to be a cheaper and time-saving screening method. The Toxic Equivalency Quotient (TEQs) determined through bio-assays can support chemical analysis in determining biologically-relevant risk assessments since bio-assay data has ecotoxicological relevance. These assays represent an integrated biological response to chemical pollutants, where biological effects are accounted for which is not possible in chemical analyses. One of the bio-assays used in the determination of the dioxin-like chemical TEQ is the H411 E reporter gene bio-assay. This assay is based on the Ah-receptor mediated toxicity of dioxin-like chemicals. Using this technique the TEQs for areas surrounding an active incinerator were determined, to indicate the distribution of these substances. The TEQs for the soil samples collected ranged between nondetectable and 154 ngTEQ/kg. There was no clear distributional pattern and the total organic carbon content in the soil did not seem to play a crucial role in the distribution of dioxin-like chemicals. Although a decrease in soil tillage showed a corresponding increase in TEQ. The predominant wind direction was taken into account but no correlation could be seen. However, meteorological parameters such as the ambient temperature and low precipitation in the area may have contributed to lower TEQ values. Cytotoxicity excluded data points and the phenomenon has to be addressed. High TEQ values in a residential area where free-range chickens are raised pose a serious concern to the level of dietary dioxin-like chemical intake. Eggs in the area could theoretically contain between 2.75 and 28.75 pgTEQ/g egg fat. Further studies are needed to determine how much dioxin-like chemicals are being transferred to humans through the consumption of free-range eggs / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

POPs are a group of chemicals that have been extensively studied over the last few years. The main reason that these chemicals have received so much scientific attention is the myriad of negative effects they have on the environment and human health. The properties that cause the deleterious effects include a high molecular stability, rendering them highly persistent. Added to this is the lipophilic and hydrophobic nature of the compounds. POPs will thus tend to bio-accumulate and bio-magnify in the environment, causing a direct threat to humans and wildlife. To address this threat, the Stockholm Convention on Persistent Organic Pollutants, under the supervision of United Nations Environment programme (UNEP), was initiated and became legally binding on 17 May 2004. All countries, including South Africa, which ratified this agreement, will be expected to monitor and regulate the formation of POPs. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are all members of the dioxin-like family of POPs. This family of chemicals pose serious health threats such as carcinogenic effects and negative effects on reproduction. These substances, with the exception of PCBs, are formed unintentionally as by-products of industrial and thermal processes. One of the main sources of dioxin-like chemicals is medical waste incinerators. In this project the area surrounding a medical waste incinerator was monitored using a bio-assay technique. The determination of dioxin concentrations is usually preformed by chemical analysis, however, bio-assays have proven themselves to be a cheaper and time-saving screening method. The Toxic Equivalency Quotient (TEQs) determined through bio-assays can support chemical analysis in determining biologically-relevant risk assessments since bio-assay data has ecotoxicological relevance. These assays represent an integrated biological response to chemical pollutants, where biological effects are accounted for which is not possible in chemical analyses. One of the bio-assays used in the determination of the dioxin-like chemical TEQ is the H411 E reporter gene bio-assay. This assay is based on the Ah-receptor mediated toxicity of dioxin-like chemicals. Using this technique the TEQs for areas surrounding an active incinerator were determined, to indicate the distribution of these substances. The TEQs for the soil samples collected ranged between nondetectable and 154 ngTEQ/kg. There was no clear distributional pattern and the total organic carbon content in the soil did not seem to play a crucial role in the distribution of dioxin-like chemicals. Although a decrease in soil tillage showed a corresponding increase in TEQ. The predominant wind direction was taken into account but no correlation could be seen. However, meteorological parameters such as the ambient temperature and low precipitation in the area may have contributed to lower TEQ values. Cytotoxicity excluded data points and the phenomenon has to be addressed. High TEQ values in a residential area where free-range chickens are raised pose a serious concern to the level of dietary dioxin-like chemical intake. Eggs in the area could theoretically contain between 2.75 and 28.75 pgTEQ/g egg fat. Further studies are needed to determine how much dioxin-like chemicals are being transferred to humans through the consumption of free-range eggs / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

Abstract Clarification of the mechanisms of cardiac-specific gene expression provides not only basic knowledge about how the gene expression is regulated in the heart, but also about the changes in the gene expression during the development of cardiovascular diseases. The purpose of this study was to analyze the mechanisms of cardiac chamber-specific gene expression and cardiac gene activation induced by mechanical load. In the present study, the experiments were carried out by using two cardiac genes, salmon cardiac peptide (sCP) and rat B-type natriuretic peptide (BNP) genes as models. sCP was discovered previously in our laboratory and turned out to be extremely cardiac-specific, representing A-type natriuretic peptide characters in an exaggerated way. In neonatal rat cardiomyocytes, the sCP promoter activity was shown to be strictly restricted to atrial cells and the promoter to be inert to cardiac hypertrophy-inducing factors. In order to find out the mechanisms of earlier proved BNP gene activation by mechanical load, BNP promoter activity was studied in vivo in adult rat hearts. The tandem GATA transcription factor binding site at position -80/-91 was shown to be essential for the BNP gene induction by angiotensin II. To clarify the possiblity to transfer the characters of the BNP gene into the sCP gene, short BNP fragments were inserted to the sCP gene promoter. The otherwise atrial-restricted sCP promoter was shown to be switched on in rat ventricular cardiomyocytes by adding a short BNP proximal promoter element to the sCP promoter, preferably near to the transcription start site. This activity was partly dependent on the -80/-91 GATA sites in the BNP promoter. Thus, A-type natriuretic peptide regulation can be switched to B-type regulation by a short proximal BNP promoter element. In conclusion, these studies reveal certain basic differences in cardiac atrial and ventricular gene expression.

No description available.