Steroid hormones belong to a group of compounds known as endocrine disruptors. They are hydrophobic compounds and are categorized as natural and synthetic estrogens. Some common household products have been implicated as estrogen mimics. Exposure effects of these compounds are felt by human and wildlife, such reproductive alterations in fish and frogs. They mainly introduced into the environment through veterinary medicines administration to animals and the discharges from wastewater treatment plants (WWTPs). In this study, a new alternative analytical procedure that is simple, rapid and fast for the determination and quantification of five steroidal hormones: estriol (E3), beta estradiol (β-E2), alpha estradiol (α-E2), testosterone (T), progesterone (P) and bisphenol A (BPA) using the High pressure liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). These compounds were studied because of their strong endocrine-disrupting effects in the environment. Under optimum conditions, a linear graph was obtained with correlation coefficient (R2) ranging from 0.9952 - 0.9996. The proposed method was applied to the analysis of water samples from a wastewater plant and the results obtained were satisfactory. The limits of detection (LOD) for the target analytes in wastewater influent was between 0.0002 – 0.0004 μg/L and the limit of quantification (LOQ) was 0.001 μg/L respectively for each of the analytes. Enrichment factors of 148- 258, and extraction efficiency 84- 102% were obtained for the target analytes; relative standard deviations (% RSD) for m = 6 were between 2.8 and 7.6%. The concentration of the EDCs in environment sample was between 0.2 - 2.3 μg/L. / Chemistry / M. Sc. (Chemistry)

<p>Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe. </p><p>Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages. </p><p>Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99. </p><p>The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls. </p><p>Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation. </p><p>A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.</p> / Dissertation

A crescente demanda por novos compostos químicos sintéticos para atender as necessidades médicas, agrícolas e industriais da população resultou no surgimento de novos contaminantes, denominados emergentes. Com a intensa utilização destes compostos em diversos produtos e o consequente aumento da concentração destes no meio ambiente, a população passou a estar exposta e sujeita aos seus possíveis efeitos deletérios. Os disruptores endócrinos fazem parte do grupo de contaminantes emergentes que tem a capacidade de interferir na ação natural dos hormônios por diferentes mecanismos, podendo agravar o quadro de doenças pré-existentes e causar diversos outros danos à saúde humana. Apesar de serem largamente utilizados na vida cotidiana, a regulamentação do uso desses compostos ainda é quase inexistente, aumentando a preocupação quanto à sua segurança. Comumente, os estudos de biomonitoramento humano para avaliar a exposição da população em geral são realizados utilizando amostras de sangue e urina. Neste sentido, o presente estudo teve como objetivo desenvolver um método alternativo para determinação simultânea de 17 compostos classificados como disruptores endócrinos: bisfenóis, parabenos, benzofenonas e triclocarban em saliva, empregando a microextração líquido-liquido dispersiva (DLLME) no preparo da amostra e cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC-MS/MS) para a determinação. Os seguintes parâmetros foram avaliados na otimização da DLLME: volume e tipo de solvente extrator e dispersor, força iônica, pH e agitação. Após a otimização, o processo de extração foi realizado adicionando-se 500 ?L de água deionizada à 500 ?L da amostra de saliva. Os solventes extrator e dispersor selecionados foram triclorometano e acetona, respectivamente (volume de 2 mL da mistura na proporção 1:3). Foi adicionada a etapa de agitação manual por dez segundos. As curvas de calibração de todos os analitos apresentaram linearidade, com coeficiente de correlação superiores a 0,99 no intervalo de concentração de 1-20 ng mL-1. O coeficiente de variação (CV) intra e interdias foram inferiores a 20% para o primeiro ponto da faixa linear e inferiores a 15% para as demais concentrações. O erro padrão relativo intra e interdias foram inferiores a 20% para o primeiro ponto da faixa linear e inferiores a 15% nas demais concentrações. Os limites de detecção (LOD) e de quantificação (LOQ) encontrados variaram de 0,01 - 0,15 ng mL-1 e 0,05 - 0,40 ng mL-1, respectivamente. Para avaliar a aplicabilidade do método, dez amostras de saliva foram analisadas e apresentaram concentrações detectáveis de bisfenol S (BPS), bisfenol AF (BPAF) e propilparabeno (PrP) ( <LOQ). O bisfenol A (BPA) e a benzofenona-3 (BP3) foram encontrados em concentrações que variaram entre 1,01-3,14 e 1,00-2,50 ng mL-1, respectivamente. O metilparabeno (MeP) foi detectado em todas as amostras, atingindo, nos níveis quantificáveis, a concentração de 17,8 ng mL-1. Portanto, o método aqui proposto é uma alternativa simples e rápida para estudos de biomonitoramento humano, capaz de determinar 17 analitos multi-classes simultaneamente com uso de baixo volume de solventes. / The growing demand of new synthetic chemical compounds to attend the medical, agricultural and industrial needs of the population has resulted in the emergence of new contaminants, known as emerging contaminants. The presence of these contaminants in several products, and its consequent concentration increase in the environment, exposed the population and subject them to possible deleterious effects. Endocrine disruptors are a group of these contaminants that have the ability to interfere at the natural action of hormones by different mechanisms, wich can worsen preexistent diseases and cause other damages to humam health. Despite the widely utilization of these compounds in everyday life, regulation of its use is almost non-existent, raising concerns about their safety. Mostly human biomonitoring studies to assess the exposure of the population are performed in urine and blood samples. In these sense, the present study aimed to develop an alternative method for simultaneous determination of 17 endocrine disrupting compounds: bisphenols, parabens, benzophenones and triclocarban in saliva, using dispersive liquid-liquid microextraction (DLLME) at sample preparation and high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) for determination. The evaluated parameters in the optimization of DLLME were: volume and type of extraction and disperser solvents, ionic strength, pH and agitation. After the optimization, the extraction procedure was performed by adding 500 ?L of deionized water to 500 ?L of saliva sample. The extraction and disperser solvents selected were trichloromethane and acetone, respectively (2 mL of the mixture in 1:3 ratio). The manual agitation for ten seconds. The matrix-matched calibration curves of all analytes were linear, with correlation coefficients higher than 0.99 in the range level of 1 - 20 ng mL-1. The intra and interday coefficient of variation (CV) was less than 20% for the first point of the linear range and less than 15% for the other concentrations. The intra and interday relative standard deviation (RSD) was less than 20% for the first point of the linear range and less than 15% for the other concentrations. The limits of detection (LOD) and quantification (LOQ) ranged from 0,01 - 0,15 ng mL-1 and 0,05 - 0,4 ng mL-1, respectively. To evaluate the applicability of the method, ten saliva samples were analyzed, showing detectable concentrations (<LOQ) of bisphenol S (BPS), bisphenol AF (BPAF) and propylparaben (PrP). Bisphenol A (BPA) and benzophenone-3 (BP3) were found in ranging concentrations from 1,01 - 3,14 and 1,00 - 2,50 ng mL-1, respectively. Methylparaben (MeP) was detected in all samples, reaching, at the quantifiable levels, the concentration of 17,8 ng mL-1. Thus, the proposed method is a simple and fast alternative for human biomonitoring studies, capable of determining 17 multi-class analytes simultaneously using low volume of solvent.

Huang, Hui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 141-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Introduction Epidemiologic data supporting the role of organochlorine pesticides in pubertal development are limited. Methods Using a nested case-control design, serum collected during pregnancy from mothers of 218 girls who reported menarche before 11.5 years of age (cases) and 230 girls who reported menarche at or after 11.5 years of age (controls) was analyzed for 9 organochlorines and metabolites. We analyzed the association between in utero organochlorine concentrations and early menarche using multivariate logistic regression controlling for mother's age at menarche, or mother's prenatal BMI. Results We did not observe an association between in utero exposure to HCB, β-HCH, ϒ-HCH, p,p′-DDT, p,p′-DDE, oxychlordane or trans-nonachlor and early menarche. Conclusions This study is the first to examine the association between in utero exposure to HCB, β-HCH, ϒ-HCH, oxychlordane or trans-nonachlor and early menarche. In utero exposure to organochlorine pesticides does not appear to have a role in the timing of menarche in this study.

novel ozone-assisted aerobic sludge digestion process to stabilize and decrease the amount of excess sludge produced during biological treatment is presented in this study. Excess sludge production is a well known burden for the treatment plants both legally and financially. Moreover, with the arise in the knowledge in recalcitrant compounds it is understood that it can act as a significant secondary pollutant. With the developed pulse ozonation method, waste activated sludge samples from Ankara Tatlar and other Wastewater Treatment Plants (WWTP) were ozonated for different periods in Erlenmeyer flasks once a day on each of four consecutive days. Flasks were continuously aerated between ozone applications on an orbital shaker. The MLVSS, MLSS, COD and OUR parameters were measured routinely during the course of four days of digestion in order to optimize the process. Also pH, CST(capillary suction time) and SVI (sludge volume index) were followed. As a result MLVSS reductions of up to 95% were achieved with an ozone dose of only 0.0056 kg O3/kg-initial MLSS, at the end of the fourth day. In another experimental set, ozone dose was increased on the last day in order to destroy the selected endocrine disrupting compounds, namely diltiazem, carbamazepine, butyl benzyl phthalate and acetaminophen and two natural hormones estrone and progesterone, which accumulated onto the sludge. Over 99% removal of these contaminants were achieved on the fourth day. The analyses were conducted by using LC(ESI) MS/MS after solid phase extraction (SPE). By this process it became possible to save on contact time, as well as achieving a bio-solids digestion far exceeding the standard aerobic process at the expense of a minimum of ozone dose with the additional micropollutants removal. The developed process is deemed superior over side-stream ozonation of activated sludge in that it does not cause any reduction in active biomass amount that should be maintained in the aeration tank.

Polybrominated diphenyl ethers (PBDEs), flame retardants, have been applied to consumer goods, such as furniture, electrical devices, textiles, and appliances for decades. Due to their physico-chemical properties, PBDEs are semi-volatile and easily leach off the consumer good during aging, stress, or normal wear and tear of the good. Once airborne, they pose an environmental health threat because they can adsorb onto dust particles, soil, or other particulates that can be inhaled, ingested, or come into contact with the dermal layer. Additionally, PBDEs have a molecular structure similar to other persistent organic pollutants, such as polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins and furans. They are a health threat due to their endocrine-disrupting nature by affecting thyroid functioning, fertility, and child development. The purpose of the study is to measure selected PBDEs in a wastewater treatment plant (WWTP) that produces reclaimed water, such that a mass balance can be completed, and to compare this mass balance with theoretically expected concentrations. The mass balance includes the collection of samples from wastewater, sewage sludge, and air at points within the WWTP. The PBDEs examined are BDE-28, 47, 99, 100, 153, 154, and 183. The second part of the study will compare effluent concentrations to reclaimed water concentrations in order to examine the potential exposure (if any) of using reclaimed water. Influent concentration of mean (sum7)PBDE was found to be 49,117 pg/L and effluent concentration was 4,603 pg/L, illustrating a 91% removal rate of PBDEs during the wastewater treatment plant. Sludge samples contained the highest total concentrations of PBDEs with mean (sum7)PBDE ranging from 14.0 to 41.3 ug/kg dry weight. Air samples were highest at the post-aeration (248 pg/m3 mean (sum7)PBDE) step due to the use of highly oxygenated air assisting in the release and volatilization of the PBDEs. Sludge was found to carry the largest mass loading at 14.2 lb/day (sum7)PBDE. Of the total mass loading of PBDEs from the WWTP, sludge is responsible for 86.7%, followed by reclaimed water and effluent (11.7% and 1.6%, respectively). The mass loading from air was negligible with less than 0.01% contribution to the total mass loading. Whereas reclaimed water overall had higher PBDE congener mean concentrations than the effluent, the independent samples t-test found no statistically significant differences between the two groups. The results of this study can be used to improve the wastewater treatment process to reduce the impact of PBDEs being released into the environment by WWTPs, and to educate the public on utilizing reclaimed water in a safe and healthy manner.

>Magister Scientiae - MSc / Tuberculosis still remains one of the world’s killer diseases. Pyrazinamide (PZA) is one of the most commonly prescribed anti-tuberculosis (anti-TB) drugs due to its ability to significantly shorten the TB treatment period. However, excess PZA in the body caused hepatotoxicity and liver damage. This, together with the resistance of the bacteria to treatment drugs, poor medication and inappropriate dosing, contribute significantly to the high incidents of TB deaths and diseases (such as liver damage). This, therefore, calls for new methods for ensuring reliable dosing of the drug, which will differ from person to person due to inter-individual differences in drug metabolism. A novel biosensor system for monitoring the metabolism of PZA was prepared with a nanocomposite of multi-walled carbon nanotubes (MWCNTs), polyaniline (PANI) and cytochrome P450 2E1 (CYP2E1) electrochemically deposited on a glassy carbon electrode (GCE). The nanocomposite biosensor system exhibited enhanced electro-activity that is attributed to the catalytic effect of the incorporated MWCNTs. The biosensor had a sensitivity of 7.80 μA/ μg mL-1 PZA and a dynamic linear range (DLR) of 4.92 – 160 ng/mL PZA. Bisphenol A (BPA) is a hormone-disrupting chemical used in production of epoxy resins and polycarbonates, which produce various products used on a daily basis. However, BPA can leach out of plastic during normal use and cause health effects such as cancer or disrupt the endocrine system. Moreover, BPA has also been proven to degrade from the containers in landfills and accumulate in groundwater and streams, thereby, polluting the environment while destroying aquatic organisms. Therefore, this also calls for new selective and sensitive methods for the monitoring of BPA. A novel biosensor system for monitoring the oxidation of BPA was prepared from a nanocomposite of polyaniline, polymethyl methacrylate and titanium dioxide nanoparticles, also electrochemically deposited on the GCE. Biosensor fabrication was conducted by immobilization of the enzyme manganese peroxidase (MnP) iii onto the nanocomposite film. The nanobiosensor also revealed enhanced electro activity, attributed to the incorporation of TiO2 nanoparticles. The biosensor system had a sensitivity of 0.3 μA/nM and a detection limit of 0.12 nM. This detection limit falls within the range of the allowed daily intake of BPA as recommended by the Food and Drug Administration (FDA, USA) and other regulatory bodies.

>Magister Scientiae - MSc / Current water treatment technologies do not remove many endocrine disruptor compounds (EDCs) such as 17α-ethynylestradiol (EE2) in its entirety, and the amount of these pollutants that continues to enter the aquatic environment through wastewater effluents is still capable of causing harmful health effects. Therefore the development of simpler and more sensitive biosensor system for detection of EE2 must be developed which have high responsiveness, low cost and easy handling. Therefore the aim of this study was to work towards the development of rapid test system of polyaniline-laccase on gold enzyme nanobiosensor (PANI-PSSA/Lac/Glu) for water estrogens. Preliminary studies were first done on the materials used in this study: estrogens, laccase, gold nanoparticles (AuNPs), and electropolymerized PANI-PSSA. Laccase was shown to be active towards EE2 and the enzyme could be stored for over three months. EE2 solution also could be used for over three months. Buffer used in this study was found to be suitable. Phosphoric acid (H3PO4) was a suitable electrolyte than hydrochloric acid (HCl) to be used for the electropolymerization of aniline and was used because it has same ions as the McIlvaine buffer (McIlB) which the post-deposition CVs indicated the formation of electrochemically very stable film. AuNPs were successfully synthesized and its size was identified to be less than 22 nm. McIlB used for testing electrochemical properties of AuNP. CVs of GC/PANI-PSSA and GC/PANIPSSA/ Au showed no difference before and after exposure to aq. EE2 solution, an indication of being re-usable and could also serve as stable immobilising platform in laccase biosensor. When interrogating with electrochemical impedance spectroscopy (EIS), the charge transfer resistance (Rct) of both GC/PANI-PSSA and GC/PANI-PSSA/Lac/Glu showed an average increase by about 2.4% and 21% before and after exposure of EE2, respectively. This shows that the GC/PANI-PSSA/Lac/Glu was a functional EE2 biosensor and showing a positive step towards achieving a re-usable biosensor for EE2 as a model water estrogen. Future work Page | vi will focus on exploring different ways of improving the biosensor’s surface regeneration and its sensitivity to EE2.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Endocrine disrupting compounds (EDCs) are exogenous compounds/chemicals which interfere with, or have adverse effects on the production, distribution and function of natural hormones, thereby affecting normal endocrine activity, health and quality of life of both humans and wildlife. The reproductive system is highly susceptible to EDCs due to it being controlled by an array of hormonal signals. The effects of EDCs on the male reproductive system include infertility, decreased sperm count, function and morphology, abnormal development of secondary sex characteristics, reproductive function and sexual behaviour as well as decreased libido. There are various sources by which EDCs enter the environment which include effluents from several industries (mining, agriculture, smelting, hazardous waste sites, manufacturing industries, etc.), sewage treatment effluents, urban and agricultural runoff and effluents which include natural and pharmaceutical chemicals excreted in the urine of humans and domestic livestock, pesticides, polychlorinated biphenyls, dioxins, plasticizers, surfactants, etc. Humans and animals can also be affected by EDCs by consuming food containing endocrine active substances. The growing concern regarding adverse effects due to EDC exposure of humans and wildlife, as well as the increased incidence of EDC contamination has prompted extensive research into the development and validation of screening tests to detect and monitor known EDCs and new substances with endocrine-disrupting capability. These screening tests involve assessing the effect of known and potential EDCs on reproductive function and development as well as hormone production. To assess the effect of EDCs on the reproductive system different methods are employed which include in vitro, in vivo and ex vivo methods. In vitro methods have been suggested as a suitable screening tool for EDC monitoring due to low costs, reduced animal usage, the use of standard and basic equipment as well as the ability to screen a large number of samples with multiple endpoints. Of the available in vitro methods, the minced testes method has been suggested as the most suitable method for screening EDCs and for this reason has been employed in this study. The aim of this study was thus to employ a minced testes method to screen samples for male reproductive toxicity using cell viability and hormone production (testosterone and estradiol) as endpoints.The first objective of this study was to optimize an in vitro testicular cell culture assay by determining both optimal luteinizing hormone (LH)&nbsp; concentration and incubation time needed for testosterone production. Testicular cell cultures were prepared and cells were treated with varying concentrations of LH (10, 1, 0.1, 0.01 and 0 mu/ml) and incubated for 4 hours and 20 hours. Testosterone production was evaluated for each incubation period. Testosterone production was significantly increased for both incubation periods at all LH concentrations tested as compared to the control. For both incubation periods, there was no significant difference in testosterone production between the different LH concentrations tested. From the data obtained, the 4 hour incubation period as well as the LH concentration of 10 mu/ml were selected as optimal for the testicular cell culture assay. The second objective of this study was to determine the effect of Tulbaghia violacea Harv. on the male reproductive system. T. violacea is a plant species indigenous to southern Africa and is used locally as a herbal remedy/medicine to treat several ailments. Cells were treated with varying concentrations of the T. violacea ethanol extract (with/without LH-treatment) and incubated for 4 hours. Hormone production and cell viability were evaluated. The results obtained from this pilot in vitro study demonstrated that the ethanol extract of T.violacea has androgenic properties by significantly increasing LH-induced testosterone production in mouse testes with no significant change in cell viability. The third objective of this study was to assess the effect of Sutherlandia frutescens(L.) R.Br and Artemisia afra Jacq. Ex Willd. on the male reproductive system. S. frutescens and A. afra are also plant species indigenous to southern Africa and used locally as a herbal remedy/medicine to treat several ailments. Ethanol extracts of each plant was prepared and cells were treated with varying concentrations of each extract (0, 156.25, 312.5, 625, 1250,2500 and 5000 &mu;g/ml) with or without LH-treatment and incubated for 4 hours. Cytotoxicity by LDH measurement and hormone production (testosterone and estradiol) were endpoints that were evaluated. The results obtained showed that the ethanol extracts of both plants are not cytotoxic to testicular cells and that A. afra decreases testosterone production at high concentrations. The fourth and final objective of this study was to assess the acute effect of four heavy metals, namely manganese, copper, cadmium and magnesium on the male reproductive system. These heavy metals are used extensively in manufacturing and mining industries. Cells were treated with varying concentrations of each metal salt (200, 100, 50, 25, 12.5, and 6.25 & mu;M) with or without LH-treatment and incubated for 4 hours. Endpoints evaluated included cell viability, testosterone and estradiol production. The results obtained showed that manganese, cadmium and copper are highly toxic to testicular cells in vitro and therefore may potentially cause reproductive toxicity. / South Africa