For the past few years there has been increasing concern surrounding a group of chemicals known as phthalate esters. In mammals, phthalates are known antiandrogens, interfering with the production or activity of testosterone. Phthalates are ubiquitous in the aquatic environment. With recent findings suggesting that antiandrogens may be responsible for much of the endocrine disruption found in wild fish populations, the study of phthalate esters has become integral to determining whether or not these anti-androgenic chemicals are of concern. I investigated whether di-n-butyl phthalate (DBP) was able to cause antiandrogenic endocrine disruption in fish under controlled laboratory conditions. Three experiments were undertaken. In the first study, two generations of fathead minnows were exposed to nominal concentrations of 6 to 100 μg/L for 21 and 150 days, respectively. The second experiment examined the effects of early life-stage exposure to DBP (50, 100 and 200 μg DBP/L) on three-spined sticklebacks. The final experiment examined the effects of DBP on adult male three-spined sticklebacks in a 21-day nesting study (15 and 35 μg DBP/L). DBP had no effect on the fecundity, survival, growth, sex ratio, or gonadal histology of the exposed fish in any of the experiments. Further, it failed to alter the expression of two steroidogenic genes in adult male sticklebacks. In contrast, DBP was often found to significantly alter plasma androgen concentrations in both species, and spiggin concentrations in the three-spined stickleback, most notably causing significantly reduced spiggin concentrations in the adult males exposed to DBP. Ultimately, DBP-exposure did not disrupt the ability of the fish to reproduce successfully, and did not appear to alter reproductive behaviours or the expression of secondary sexual characteristics. In conclusion, while DBP did appear to have some capacity for endocrine disruption in fish, it was unable to interfere with the ability of the fish to develop normally and reproduce successfully. Thus, environmentallyrelevant concentrations of phthalate esters are likely not of particular concern to fish populations.

A number of widely used industrial chemicals have been shown to possess endocrine-disrupting properties. In this thesis, a series of in vitro tests, and an in vivo reproductive performance test with fathead minnows, were used to clarify the extent of estrogenic activity exhibited by the phthalate esters - a class of compound hitherto referred to as 'estrogenic'. Using a recombinant yeast estrogen screen, I demonstrated that a small number of commercially available phthalates showed extremely weak estrogenic activity. The most potently estrogenic phthalate of those tested was BBP, which was approximately one million-fold less potent than 17B-estradiol. The phthalates which were estrogenic in the yeast screen were also mitogenic on estrogen-responsive human breast cancer cells (MCF-7 and ZR-75 cell lines). The most prolifically used phthalate, DEHP, was not estrogenic in any of these assays. The small number of metabolites of phthalate metabolites tested (including MBuP, MBzP, MEHP and MnOP) were also not estrogenic in the recombinant yeast assay. The ability of BBP (as the most potently estrogenic phthalate in vitro) to induce a vitellogenic response (an indicator of estrogen exposure) in fathead minnows (Pimephales promelas) exposed via the water was assessed. No induction of vitellogenin was observed, indicating that 100 Jlg BBP/L (a concentration higher than would normally be found in the environment) is not estrogenic to this species of fish under the conditions employed for this experiment. In the same study, fecundity of breeding pairs of fathead minnows was assessed; exposure to BBP was not found to affect reproductive performance in these fish. A possible alternative mechanism of action of the way in which the phthalates induce frequently reported reproductive disorders was observed. Some of the phthalates, and, notably, some of their metabolites, were demonstrated to act as anti-androgens in a recombinant yeast androgen assay. 4-Nonylphenol is another industrial chemical which is used in large volumes, and due to the nature of its use (mainly in detergent formulations), is discharged into water systems via sewage effluents. This chemical has been shown to be estrogenic to fish at the concentrations at which it has been detected in the environment. 4-Nonylphenol was tested for its ability to affect plasma and pituitary gonadotropin levels in female recrudescing rainbow trout (Oncorhynchus mykiss). Plasma and pituitary levels of FSH were suppressed in fish exposed to 10 and 100 Jlg 4-NP/L. In addition, FSH gene expression was reduced in these fish, and also in the fish exposed to 1 Jlg 4-NP/L. Pituitary LH content and gene expression of this hormone were suppressed in the fish exposed to 100-, and 10- and 100 Jlg 4-NP/L respectively. Gonadal development in vertebrates is regulated by FSH. Ovarian development ceased in the fish exposed to 100 JAg 4-NP/L, possibly as a result of the suppression of FSH synthesis and/or release in these fish.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Endocrine disrupting compounds (EDCs) are exogenous compounds/chemicals in the environment that interfere with the synthesis, secretion, distribution and function or elimination of natural hormones in the body. Environmental estrogens are a subclass of EDCs that may mimic or inhibit the effect of endogenous estrogen and can therefore influence developmental and reproductive health in humans and animals. EDCs have been reported to adversely affect the reproductive, immune, endocrine and nervous systems of wildlife and humans. The effects of EDCs include gonadal abnormalities, altered male/female sex ratios, reduced fertility and cancers of the male and female reproductive tract to mention a few. These effects are difficult to detect. Although it is essential to screen for EDCs in aqueous environmental samples, most countries have failed to implement this as part of their routine water quality monitoring programs due to various constraints such as the high cost of assays and the lack of infrastructure and skills required to do the assays. Therefore, there is a clear need for more user-friendly, more economically viable and time saving assays that can be used for routine monitoring of environmental EDCs. The aim of this study was to investigate the comparison between chemical and tissue culture methods to monitor environmental estrogens. 28 environmental water samples were collected from various sites around South Africa and analyzed for EDCs using a battery of rapid in vitro tests. Samples collected for the current study were selected based on various human impacts and also to give approximately 50% high and 50% low estrogen values. The 28 environmental water samples were separated into two groups based on the estradiol ELISA. The estradiol ELISA was chosen because estradiol is the principal estrogen found in all mammalian species during their reproductive years. For this separation, an estradiol level of 5 pg/ml was used as cut-off. Of the 28 samples investigated, 15 had estradiol levels higher than 5 pg/ml and were designated as high estradiol. The remaining 13 samples contained estradiol at 5 pg/ml or less and they were designated as low estradiol The first objective of this study was to compare different rapid ELISAs for EDC monitoring to determine if the data obtained with these assays are similar/identical. The data obtained from the estrogenic ELISAs was related/similar and showed good correlation with each other. This is because the different estrogens are very similar and also due to the fact that the same sub-group in the population (the reproductively active females) is secreting these hormones. Therefore, an estradiol rapid assay was proposed as a first screening system for estrogens in samples. Even though there was a positive correlation between the estradiol rapid assay and testosterone rapid assay, separation of samples based on estradiol levels wasn’t a good predictor of testosterone levels in the samples. A testosterone rapid assay was therefore recommended as necessary to screen for androgens in samples. The positive correlation between the estradiol rapid assay and progesterone rapid assay was expected because both estradiol and progesterone are secreted and excreted by the same population sub-group (reproductively active females). This study also demonstrated a good predictability of separating samples containing progesterone using the estradiol ELISA. Progesterone is secreted by pregnant women, a sub-group of the reproductively active females. It is advised that a progesterone rapid assay be included to screen samples for progestogens The second objective of this study was to compare estradiol rapid ELISAs with a bioassay for anti-androgenicity using mouse testicular cell cultures. The mouse testicular cell testosterone synthesis bioassay to monitor anti-androgenicity of the samples showed no correlation between the ELISA data for estrogens. This study shows that anti-androgenic effects need to be monitored independently because the data for estrogenic compounds cannot be used as a predictor for anti-androgenic effects. This demonstrated the need for the inclusion of a mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity of water samples. In summary, due to the different mechanisms of action of EDCs, this study recommended a battery of assays to monitor for EDCs. The battery of assays suggested is: v progesterone rapid assay was expected because both estradiol and progesterone are secreted and excreted by the same population sub-group (reproductively active females). This study also demonstrated a good predictability of separating samples containing progesterone using the estradiol ELISA. Progesterone is secreted by pregnant women, a sub-group of the reproductively active females. It is advised that a progesterone rapid assay be included to screen samples for progestogens. The second objective of this study was to compare estradiol rapid ELISAs with a bioassay for anti-androgenicity using mouse testicular cell cultures. The mouse testicular cell testosterone synthesis bioassay to monitor anti-androgenicity of the samples showed no correlation between the ELISA data for estrogens. This study shows that anti-androgenic effects need to be monitored independently because the data for estrogenic compounds cannot be used as a predictor for anti-androgenic effects. This demonstrated the need for the inclusion of a mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity of water samples. In summary, due to the different mechanisms of action of EDCs, this study recommended a battery of assays to monitor for EDCs. The battery of assays suggested is: Estradiol ELISA as a rapid assay to screen for estrogens. Testosterone ELISA as a rapid assay to screen for androgens. Progesterone ELISA as a rapid assay to screen for progestogens. Mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity.