Reporter genes are often used to observe expression of promoters, which may change from its natural behaviour as a result of stress or disease states. Reporter genes are useful because they are easily detectable by a variety of imaging methods, including fluorescence microscopy techniques, magnetic resonance imaging, and positron emission tomography. Previously, methyl 5-MeO-N-aminoanthranilate (MMNA) had been synthesized as an aldehyde-conditional fluorophore and was tested in physiological conditions to identify the Aldehydic Load of cells. Thus, it was hypothesized that a reporter protein displaying an aldehyde on the cell surface can be identified by MMNA. This reporter protein would contain a substrate recognition site for formylglycine generating enzyme (FGE) that converts a specific cysteine residue into a formylglycine residue. This will result in production of an aldehyde at the N-terminal of the transmembrane domain of platelet derived growth factor receptor . In this way, the protein product, Aldehyde-presenting FGE-dependent Readout (Alfred), would display an aldehyde on the extracellular surface of the cell. Alfred was expressed in A549 human lung cancer cells using the Tet-On® Inducible System, which allows expression of a gene of interest by use of doxycyclin (dox) as a chemical trigger. Microscopy of Alfred-transfected cells, induced by dox and probed with MMNA, showed no difference in fluorescence between non-transfected and Alfred-transfected cells. The overexpression of FGE to increase thiol-to-aldehyde conversion, and the imaging of cells at longer timepoints (48 and 72 hours) to allow localization of the protein to the cell surface, were attempted. In addition, Alfred was constitutively expressed in another transfection experiment in efforts to increase gene expression. However, these efforts to evaluate Alfred did not improve the microscopy results. Western blotting confirmed FGE overexpression in transgenic cells. Blotting against the Myc-tag in Alfred showed no detected proteins in Alfred-transfected cells. In conjunction with the microscopy images, these results suggest that Alfred is not expressed and cannot be detected as a reporter gene. Comparison to previous works allows the identification of potential approaches to improve Alfred functionality, including the absence of the hemagglutinin epitope, the choice of aldehyde probe used, the choice of cell line used, and the method of analyzing microscopy. Future directives are postulated to identify sources that hinder Alfred expression, and to improve visualization of Alfred over homeostatic aldehydes.

Biologiska läkemedel är stora molekyler som har renats eller producerats från ett biologiskt ursprung. De används idag i stor utsträckning runt om i världen på grund av deras effektivitet att minska symtomen som kan uppstå vid olika mycket svåra sjukdomar. Detta arbete fokuserar på biologiska läkemedel som hämmar cytokinet tumörnekrotisk faktor (TNF). Läkemedlen används bland annat vid svåra autoimmuna sjukdomar. Vid autoimmuna sjukdomar aktiveras cellerna i immunsystemet vilket leder till utsöndring av olika cytokiner till exempel interleukin 1 och TNF. Dessa cytokiner påverkar kroppen på olika sätt genom att aktivera eller inhibera vissa produktionsprocesser i kroppen. Syftet med projektet var att verifiera en cellbaserad metod som används för koncentrationsmätning av det biologiska läkemedlet Infliximab (en TNF antagonist) i patientserum. Metoden som används i detta projekt kallas för reporter gene assay och den baseras på användning av en iLite cellinje. iLite är humana erytroleukemiceller K562 som är transfekterade med nukleär faktor kappa-B. Denna faktor aktiveras när TNF binder sig till TNF receptorer på cellytan. Aktiveringen av denna faktor leder till produktion av enzymet Eldflugeluciferas. Produktionen av enzymet är indirekt proportionell mot koncentrationen av TNF antagonist i serum. Dessutom innehåller cellerna en reporter gen för Renilla luciferas som uttrycks konstant. Renilla luciferas används som en internkontroll för att normalisera variationer som kan orsakas av provhantering samt transfektionseffektivitet. Luminescensen som utvecklas på grund av inverkan av Eldflugeluciferas på luciferas substrat samt inverkan av Renilla luciferas på stopp substrat mäts med hjälp av en luminometer. Dag-till-dag variationen för fem analyserade prov som analyserades vid fyra olika tillfällen var mellan 5 - 28 %, lot-till-lot variationen för fyra prov som analyserades fyra gånger vid fyra olika tillfällen var mellan 14 - 21 %. Alla normala serumprover från friska frivilliga blodgivare var negativa med en koncentration av Infliximab mindre än 0,65 µg/ ml. Resultaten visar att metoden är stabil och känslig jämfört med Biomonitors resultat. Dessutom visar resultaten att medelvärden av de olika körningarna från Euro Diagnostica (ett laboratorium som tillverkar immunologiska kit, Malmö) korrelerar väl med resultaten från Biomonitors laboratorium i Köpenhamn. Korrelationen för 19 analyserade prov var 0,99. / Biological drugs are large molecules that have been purified from or produced from a biological origin. They are used in great extent around the whole world because of their efficiency in reducing the symptoms of very severe diseases. The present work focus on biological drugs that inhibit tumor necrosis factor (TNF). Biological drugs are used for treatment of different autoimmune diseases. In autoimmune diseases activated cells of the immune system leads to secretion of various cytokines such as interleukin 1 and TNF. These cytokines affect the body in different ways by activating or inhibiting certain processes in the body. The purpose of this project was to verify a cell-based method which used for concentration measurement of the biological drug Infliximab (a TNF antagonist) in patient serum. The method which used in this project is called the reporter gene assay, and it is based on the use of an iLite cell line. iLite are human erythroleukemia K562 cells which are transfected with the nuclear factor kappa-B. This factor is activated when the tumor necrosis factor binds to its receptors on the cell surface. The activation of this factor leads to the production of the enzyme Firefly luciferase. Production of the enzyme is indirectly proportional to the concentration of TNF antagonist in the serum. Moreover, the cells contain a reporter gene for Renilla luciferase which is under constant expression. Renilla luciferase is used as an internal control to normalize the variations that may be caused by sample handling and transfection efficiency. The luminescence developed due to the influence of Firefly luciferase on luciferase substrate and Renilla luciferase on stop substrate is measured by using a luminometer. Day-to-day variations for five samples were analyzed at four different times were between 5 - 28 % while variations of lot-to-lot of 4 samples were analyzed at four different times were 14 - 21 %. The concentration of Infliximab in all the serum samples from healthy volunteer blood donors were less than 0.65 µg / ml. The results show that the method is robust and sensitive as compared to Biomonitors results. Furthermore, the results show that the mean value of the different runs from Euro Diagnostica (a laboratory that produces immunological kits, Malmö) for day-to-day and lot-to-lot variation agrees quite well with those from Biomonitors laboratory in Copenhagen. The correlation for the nineteen analyzed samples was 0.99.

No description available.

No description available.

A non-replicating recombinant adenovirus, Ad5MCMVlacZ, which expresses the 13-galactosidase (l3-gal) reporter gene, was used to examine both constitutive and inducible repair of UVC-damaged DNA in Chinese hamster ovary (CHO) cells. Host cell reactivation (HCR) of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated nucleotide excision repair (NER) proficient parental CHO-AA8 and m mutant CHO-UV61 cells which are deficient in the transcription-coupled repair (TCR) pathway of NER. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for 13-gal activity 24 h later. HCR of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was significantly reduced in non-irradiated CHO-.UV61 cells compared to that in non-irradiated CHO-AA8 cells suggesting that repair in the transcribed strand of the UVC-damaged reporter gene in untreated CHO-AA8 cells utilizes TCR. Prior UVC-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR for expression of the UVC-damaged reporter gene in CHO-AA8 cells but not m TCR deficient CHO-UV61 cells. Pre-UVC-treatment of cells resulted also in an enhanced expression of 13 -gal for unirradiated Ad5MCMVlacZ in both CHO-AA8 and CHO-UV61 cells. However, compared to CHO-AA8 cells, the CHO-UV61 cells exhibited comparable levels of enhanced 13-gal activity following significantly lower UVC exposures to cells suggesting that persistent damage in active genes plays a direct role in enhancing 13-gal activity driven by the MCMV promoter in CHO cells. These results suggest that prior UVC treatment results in a transient enhancement in repair of UVC-damage DNA in the transcribed strand of the active reporter gene in CHO-AA8 cells through an enhancement of TCR or a mechanism that involves the TCR pathway and that the upregulation of reporter gene expression alone is not sufficient for enhanced repair of the reporter gene in CHO-UV61 cells. The HCR assay was used also to examine both constitutive and inducible repair of UVC-damaged DNA in mouse embryonic fibroblast (MEF) cells. HCR of B-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated NER proficient parental wild type MEF cells and in MEF cells with specific knockouts in the p53 (p53-/-), pRb (pRb-/-), and p107 (p107-/-) genes. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for ~-gal activity 24 h later. HCR of ~-gal activity for UVC-irradiated Ad5MCMVlacZ did not show a significant difference in non-irradiated cells for any of the MEF knockouts cells compared to the parental strain suggesting that p53, pRb and p107 does not play a role in repair of the UV -damaged reporter gene in untreated MEF cells. Prior UVC-irradiation of cells with low UVC fluences resulted in an enhancement of HCR for expression of the UV C-damaged reporter gene in MEF wild type cells, low passage pRb-/-and p 1 07 -I-MEF cells but not in p53-/-MEF cells or in high passage pRb-/-and p107-/-MEF cells. These results suggest that prior UVC treatment MEF cells results in an induced repair of UVC-damaged DNA that is dependent on p53. The presence of an enhancement of HCR for the UVC-damaged reporter gene in pre-UVC treated cells in low passage, but not in high passage, pRb-/-and p 1 07-I-cells suggests that the lack of pRb or pI 07 expression per-se does not result in a deficiency in inducible DNA repair. However, these results suggest that the lack of pRb or p 1 07 expression results in alterations in MEF cells at high passage number that abrogate inducible repair of UVC-damaged DNA. UVA produces predominantly single base damage that is repaired through base excision repair (BER), whereas UVC and UVB produce predominantly cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PP) that are repaired through NER. The colony survival following exposure to various UV sources was examined in cells proficient and deficient in (NER). The UV sources were a UVC source from a germicidal lamp emitting predominantly at 254 nm. and a UVA source from a lKW Hg-Xe arc lamp using either a Band pass filter (BPF) or a 335 Cut-off-filter (335COF). NER deficient CHO-UV5 and CHO-UV61 cells were more sensitive to UVC exposure compared to NER proficient CHO-AA8 cells, consistent with the production of UVC-induced DNA damage predominantly in the form of CPDs and 6-4PPs which are repaired through the NER pathway. NER deficient xeroderma pigmentosum cells from complementation group D (XPD) were more sensitive compared to NER proficient normal human cells following exposure to the UVA-BPF source. In addition XPDdenV cells, which express the denY gene from bacteriophage T4, were more resistant than XPD cells following exposure to the UVA-BPF source. Since the denY protein is specific for excision ofCPDs these results indicate a substantial proportion of the induced DNA damage resulting from the UV A-BPF is in the form of CPDs, presumably due to a significant UVB component in the beam. In contrast, the NER deficient CHO-UV5 and CHO-UV61 cells showed a similar sensitivity compared to the NER proficient CHO-AA8 cell line following UVA-335COF exposures up to 60 KJ/m2• However, for UVA-335COF exposures greater than 60 KJ/m2 the NER deficient cells were more sensitive compared to the NER proficient CHO-AA8 cells, although the difference in sensitivity between NER deficient and NER proficient cells was less than that detected following UV A-BPF exposure. These results suggest that the UVA-335COF exposure produces predominantly DNA damage of the single base type for exposures less 60 KJ/m2. This is consistent with the calculated spectral distribution, which showed a 5.62% UVB component for the UVA-BPF, but only 0.14% UVB component for the UVA-335COF. / Thesis / Master of Science (MS)

Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen. / The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.

No description available.

South Africa ratified the Stockholm Convention (SC), which became legally binding on 17 May 2004. This Convention targets 12 particularly toxic persistent organic pollutants (POPs) for virtual elimination. The Convention also requires parties to reduce the release of organochlorine pesticides and the intentionally- and unintentionally-produced POPs such as dioxins, furans and polychlorinated biphenyls (PCBs) (referred to as dioxin-like chemicals). Dioxins are a heterogeneous mixture of chlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) congeners. These substances were never intentionally produced but are produced as by-products of industrial processes (such as metallurgical processes and bleaching of paper pulp). They can also be formed during natural processes such as volcanic eruptions and forest fires. The largest contributor to releases of PCDD/Fs in the environment is incomplete combustion from waste incinerators leading to the unintentional production of these compounds. Polychlorinated biphenyls (PCBs) are used in transformers and capacitors, but can also be formed unintentionally during industrial and thermal processes. Dioxin-like chemicals (PCDD/Fs and/or PCBs) are classified as persistent because of the following characteristics: lipophilicity and hydrophobicity; resistance to photolytic, chemical and biological degradation and they are able to travel long distances. As South Africa is a semiarid region, POPs will be less prone to travel here because these substances favour colder regions with high soil organic matter. Fish, predatory birds, mammals (including humans) absorb high concentrations of POPs through the process of bio-concentration, leading to bio-accumulation of these substances in the fatty tissue. PCDD/Fs occur as unwanted trace contaminants in air, water, land, in residues and products (such as consumer goods e.g. paper and textiles). The distribution of these chemicals into various matrices is problematic since they cause damage to the environment and human health. These chemicals pose a threat to human health when found in high concentrations that may lead to acute hepatoxicity and dermal toxicity (chloracne). Long-term exposure to low concentrations of these substances might lead to chronic effects such as reproductive problems and carcinogenicity. Since ferrous and non-ferrous metal production is a source of dioxin-like chemicals, a platinum mine in the Limpopo Province, South Africa, was selected for this investigation. The aim of the study was to determine if there are dioxin-like chemicals associated with platinum mining and processing, and if the H4IIE reporter gene bio-assay could be used to semi-quantify and assess the potencies of the complex environmental and process samples by determining their Toxic Equivalency Quotients (TEQ). The implications of the sources to the formation of dioxin-like chemicals regarding the SC were investigated and recommendations were made to improve this study. Samples were collected from tailings dams, woodchips, a dumpsite and slag from the smelter at Union Section. Samples were extracted with the Soxhlet apparatus using hexane as solvent. The percentage total organic carbon (%TOC) was determined for each sample to normalise the data. The method used was the Walkley-Black method. In determining the TEQ of each sample, the H4IIE luc cell line was used. The cells of the H4IIE luc line are genetically modified rat hepatoma cells stably transfected with a luciferase firefly gene. The luciferase gene is activated by the presence of dioxin-like compounds and the concentration of the enzyme is measured as relative light units (RLUs). The amount of RLUs is directly proportional to the dioxin load in the extract. This method is rapid, cost and time-effective in determining the TEQ when compared to chemical analysis. The TEQ2o-valuesin the various samples, as determined with the H4IIE luc cell line, ranged from 0.007 ngTEQ/kg to 54.06 ngTEQ/kg. Thermal processes at the smelter, sorption of hydrophobic organic compounds (HOCs) to soil and tailings, and external sources such as anthropogenic activities contributed to high TEQ2o-values. Climatic conditions, wind, precipitation, and solubility of HOCs into surfactants lead to low TEQ20. The smelter at Union Section had a very high TEQ20of 44.62 ngTEQ/kg compared to Impala Platinum mine (5.15 ngTEQ/kg). This implies that workers at Union Section are possibly exposed to low and high concentrations of dioxin-like chemicals. Long-term exposure to these compounds could lead to bio-accumulation in the fatty tissue of the mine workers, leading to chronic effects such as reproductive problems and cancer. The air emission of the furnace at the smelter was 0.03 gTEQ/annum and the release of the PCDD/Fs into the slag was 0.60 gTEQ/annum. By effectively managing the smelter it is possible to reduce the TEQ. The TEQ of each sample increased due to normalising the data. The normalised TEQ20 ranged from 0.94 ng TEQ/kg to 42497.48 ngTEQ/kg. Dioxin-like chemicals are present on a platinum mine, but at varying quantities and the effects of these compounds might be detrimental to the environment and the workers at the platinum mine. Further analyses of the health impacts associated with the platinum mine are needed. The H4IIE reporter gene bio-assay could be used to effectively determine the TEQ of each sample. Although this investigation has identified the formation and presence of dioxin-like chemicals at certain stages of mining and processing, not all of the processes were investigated. Some of these processes have the potential to add, and even destroy, these chemicals, affecting potential human exposure and amounts released to the environment. This, however, requires further investigation. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

Thesis (M. Environmental Science (Water Science))--North-West University, Potchefstroom Campus, 2007.

South Africa ratified the Stockholm Convention (SC), which became legally binding on 17 May 2004. This Convention targets 12 particularly toxic persistent organic pollutants (POPs) for virtual elimination. The Convention also requires parties to reduce the release of organochlorine pesticides and the intentionally- and unintentionally-produced POPs such as dioxins, furans and polychlorinated biphenyls (PCBs) (referred to as dioxin-like chemicals). Dioxins are a heterogeneous mixture of chlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) congeners. These substances were never intentionally produced but are produced as by-products of industrial processes (such as metallurgical processes and bleaching of paper pulp). They can also be formed during natural processes such as volcanic eruptions and forest fires. The largest contributor to releases of PCDD/Fs in the environment is incomplete combustion from waste incinerators leading to the unintentional production of these compounds. Polychlorinated biphenyls (PCBs) are used in transformers and capacitors, but can also be formed unintentionally during industrial and thermal processes. Dioxin-like chemicals (PCDD/Fs and/or PCBs) are classified as persistent because of the following characteristics: lipophilicity and hydrophobicity; resistance to photolytic, chemical and biological degradation and they are able to travel long distances. As South Africa is a semiarid region, POPs will be less prone to travel here because these substances favour colder regions with high soil organic matter. Fish, predatory birds, mammals (including humans) absorb high concentrations of POPs through the process of bio-concentration, leading to bio-accumulation of these substances in the fatty tissue. PCDD/Fs occur as unwanted trace contaminants in air, water, land, in residues and products (such as consumer goods e.g. paper and textiles). The distribution of these chemicals into various matrices is problematic since they cause damage to the environment and human health. These chemicals pose a threat to human health when found in high concentrations that may lead to acute hepatoxicity and dermal toxicity (chloracne). Long-term exposure to low concentrations of these substances might lead to chronic effects such as reproductive problems and carcinogenicity. Since ferrous and non-ferrous metal production is a source of dioxin-like chemicals, a platinum mine in the Limpopo Province, South Africa, was selected for this investigation. The aim of the study was to determine if there are dioxin-like chemicals associated with platinum mining and processing, and if the H4IIE reporter gene bio-assay could be used to semi-quantify and assess the potencies of the complex environmental and process samples by determining their Toxic Equivalency Quotients (TEQ). The implications of the sources to the formation of dioxin-like chemicals regarding the SC were investigated and recommendations were made to improve this study. Samples were collected from tailings dams, woodchips, a dumpsite and slag from the smelter at Union Section. Samples were extracted with the Soxhlet apparatus using hexane as solvent. The percentage total organic carbon (%TOC) was determined for each sample to normalise the data. The method used was the Walkley-Black method. In determining the TEQ of each sample, the H4IIE luc cell line was used. The cells of the H4IIE luc line are genetically modified rat hepatoma cells stably transfected with a luciferase firefly gene. The luciferase gene is activated by the presence of dioxin-like compounds and the concentration of the enzyme is measured as relative light units (RLUs). The amount of RLUs is directly proportional to the dioxin load in the extract. This method is rapid, cost and time-effective in determining the TEQ when compared to chemical analysis. The TEQ2o-valuesin the various samples, as determined with the H4IIE luc cell line, ranged from 0.007 ngTEQ/kg to 54.06 ngTEQ/kg. Thermal processes at the smelter, sorption of hydrophobic organic compounds (HOCs) to soil and tailings, and external sources such as anthropogenic activities contributed to high TEQ2o-values. Climatic conditions, wind, precipitation, and solubility of HOCs into surfactants lead to low TEQ20. The smelter at Union Section had a very high TEQ20of 44.62 ngTEQ/kg compared to Impala Platinum mine (5.15 ngTEQ/kg). This implies that workers at Union Section are possibly exposed to low and high concentrations of dioxin-like chemicals. Long-term exposure to these compounds could lead to bio-accumulation in the fatty tissue of the mine workers, leading to chronic effects such as reproductive problems and cancer. The air emission of the furnace at the smelter was 0.03 gTEQ/annum and the release of the PCDD/Fs into the slag was 0.60 gTEQ/annum. By effectively managing the smelter it is possible to reduce the TEQ. The TEQ of each sample increased due to normalising the data. The normalised TEQ20 ranged from 0.94 ng TEQ/kg to 42497.48 ngTEQ/kg. Dioxin-like chemicals are present on a platinum mine, but at varying quantities and the effects of these compounds might be detrimental to the environment and the workers at the platinum mine. Further analyses of the health impacts associated with the platinum mine are needed. The H4IIE reporter gene bio-assay could be used to effectively determine the TEQ of each sample. Although this investigation has identified the formation and presence of dioxin-like chemicals at certain stages of mining and processing, not all of the processes were investigated. Some of these processes have the potential to add, and even destroy, these chemicals, affecting potential human exposure and amounts released to the environment. This, however, requires further investigation. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.