|dc.description.abstract||Sex steroid production needs to be maintained within the physiological set point range, deviations outside of the normal range can be a symptom of- and risk factor for endocrine-related diseases (such as polycystic ovarian syndrome (PCOS), hypogonadism, breast- and prostate cancer). Testosterone is the dominant male sex steroid and estradiol (E2) is the most abundant and potent female sex steroid. Testosterone and E2 are mainly synthesised in gonads of both sexes. Testosterone and E2 induce a cellular response by diffusing into cells and binding to their respective nuclear receptor (androgen- (AR) and estrogen receptor (ER)), which upon activation, operate as transcription factors and regulators of gene transcription. It is clinically important to have an accurate measure of detection for testosterone and E2. The current clinical measures, which are immunoassay-based techniques, are under scrutiny due to their poor sensitivity and reproducibility, especially at low concentrations. Furthermore, although testosterone and E2 are the most prominent endogenously synthesised sex steroids, they are not the only molecules capable of activating their intracellular cognate receptor. Other endogenously synthesised androgenic and estrogenic precursor molecules to testosterone and E2 can also activate AR and ER, respectively. Furthermore, studies have shown that exogenous (anti-) estrogens and androgens sourced from synthetic and plant origin have potential to activate or inhibit AR and ERs.
A novel technique to identify all estrogenic and androgenic molecules within a sample is reporter gene bioassays (RGBs). RGBs are a cell-based technique that can measure the level of ER or AR activation by a test substance. Cells can be transfected with an expression plasmid to over express AR or ER/ and a reporter gene plasmid that harbours the response element specific to the receptor, which is upstream of a reporter gene. Alternatively, a cell line with endogenous AR or ER/ is transfected with a reporter gene plasmid only. Upon activation of AR or ER/, the receptor binds to its response element on the reporter plasmid to initiate gene transcription of the reporter gene, which is translated into an enzyme, such as luciferase. The reporter gene is measured following addition of a substrate to quantify the level of light emission, which is proportional to the level of receptor activation. Via this mechanism, all molecules that are capable of activating the receptor can be monitored.
The overarching aim of this study was to determine if RGBs can be used to measure clinically relevant samples. ER- and AR-RGBs hosted in T47D and HEK293 cells, respectively, were optimised to measure serum and plasma samples. The net serum estrogenicity and androgenicity measurement was compared with the data of the clinical standard immunoassay to determine if additional information could be provided. A major part of this PhD was the establishment of a clinical study, whereby serum from post-menopausal females was obtained for the purpose of measuring in the ER- and AR-RGBs. All patients recruited to the study started letrozole treatment and the aim of this clinical study was to determine if changes in ER- and AR bioactivity could be measured after the onset of treatment. Letrozole is an aromatase inhibitor that blocks the conversion of androgens into estrogens. Therefore, both ER- and AR-RGBs were utilised in a clinical setting to determine their clinical efficacy by comparing to the results of testosterone and E2 immunoassay data.
Overall, both ER- and AR-RGBs provided a readout for all serum and plasma samples tested, whereas only some samples had a detectable level of testosterone and/or E2 by immunoassay. This study shows that information to companion quantitative measures of E2 and testosterone can be derived from ER- and AR-RGBs.||