The development and implementation of biomarker assays for estrogenic endocrine disruptors

Abstract

Endocrine disrupting chemicals (EDCs) are compounds found in the environment that have the potential to disrupt normal endocrine function. Estrogenic EDCs (e-EDCs) is a subclass of EDCs and is defined as substances contaminating the environment that may mimic or inhibit the effect of endogenous estrogen and therefore may influence developmental and reproductive health in humans and animals. The aim of this study was to develop, validate and implement a battery of in vitro and in vivo screening assays for e-EDCs. The first objective of this study was the validation of commercially available Enzyme linked Immunosorbent Assay (ELISA) kits (designed for hormone quantification in human serum) to detect natural estrogenic hormones. These ELISA kits were validated to quantify estrone, estriol and 17β-estradiol (E2) in complex environmental water extracts. These ELISAs were proved to be very sensitive and reliable with detection limits of 0.3 ng/ml, 15 pg/ml and 25 pg/ml for estriol, estrone and E2 respectively. The ELISAs showed inter- and intra assay variations of less than 10 %. The second objective was the development of a multi biomarker whole cell bioassay that can be used as a rapid, sensitive and inexpensive screen for total environmental estrogenic activity as well as cytotoxicity. The human breast cancer cell line, MCF-7 was used to develop this assay. Total lactate dehydrogenase (LDH) activity, total protein yield and mitochondrial activity (XTT activity) were evaluated as proliferation biomarkers for estrogenic endocrine disruption. The current study shows that the total cellular LDH assay is the most sensitive assay for E2 dependent proliferation monitoring. The MCF-7 total LDH assay can be used to detect estrogens over the range from 0.1-1000 nM E2. It was also showed that the well know E-screen assay can be performed in culture medium containing serum replacement factor instead of fetal bovine serum. The third objective of this study was to develop a direct ELISA on cultured MCF-7 cells to quantify ERα levels as a biomarker for estrogenicity. This biomarker had a detection range between 0.1 - 1000 nM for E2. ERα levels were suppressed in the presence of estrogen. Results obtained with the ERα ELISA for estrogenicity showed a good correlation with the total cellular LDH assay for estrogenicity in MCF-7 cells. This ELISA was successfully employed to assess environmental water extracts for estrogenicity. The forth objective of this study was to setup and validate a competition ELISA to quantify vitellogenin (VTG) from Oreochromis mossambicus (tilapia). A competition ELISA for tilapia VTG using commercially available antibodies was developed. This tilapia VTG competition ELISA has a broad detection range between 80 ng/ml – 5.4 µg/ml VTG and is able to detect in vitro and in vivo synthesized VTG. The fifth objective of the study entailed using this quantitative ELISA to validate the use of tilapia juveniles (less than 70 days post fertilization (DPF)) as a short term in vivo screen for estrogenicity. Tilapia juveniles between 35 and 45 (DPF) exhibited the maximum ability to induce VTG upon estrogen exposure. At this age, 200 ng/l of diethylstilbestrol (DES) was the lowest concentration of estrogen that was able to induce significant VTG levels after 10 days of exposure. Analysis of the natural VTG levels of a mixed sex population of juveniles at 5 day intervals between 15 and 70 DPF showed a peak at 50 DPF which coincided with a peak in natural whole body homogenate E2 concentrations. This study was concluded by implementing this battery of assays to assess the Eerste River, South Africa at three sampling sites, namely Jonkershoek, Stellenbosch sewage treatment works (STW) effluent and Spier for e-EDCs. The control site, Jonkershoek contained very low levels of estrone. Water from this site showed no estrogenic activity when the E-screen and the ERα induction in MCF-7 cells. Some of the water samples collected at this site tested positive for estrogenicity when analysed with the juvenile tilapia VTG assay, whereas the rest were negative. The estrone levels in the sewage effluent extracts as well as Spier were significantly higher. The assay using ERα protein induction by the MCF-7 cell line, the MCF7 proliferation assay and the tilapia in vivo screen for estrogenicity showed that these samples are estrogenic. Results obtained for estrogenicity at the three different sampling sites for each of the assays in the battery were comparable. In this study we developed, validated and also implemented a battery of assays encompassing both in vitro and in vivo assays, based on different biological mechanisms, to detect estrogenic EDCs. To our knowledge, this is the first study that has used a battery of bioassays to specifically assess a South Africa river for estrogenicity.