Endocrine disrupting chemicals that enter the environment have broad consequences on human health. Humans and fish share similarities in genes, development, and the HPG axis. Fish also provide observable endpoints of reproduction. As a result in vivo tests with model fish, such as the zebrafish, are an integral component to assess the health effects of these chemicals in humans. Circulating steroid hormones are critical biomarkers of endocrine disruption, but measurements of steroids are challenging in zebrafish because of the limited (<5 uL) blood volume, laborious sample processing, and the number of animals investigated. The proposed activities are based on capillary electrophoresis and create a high-throughput, label-free method to assay steroids in plasma. Two analytical methods are developed to support a systematic approach to rapidly profile steroids. Steroid biomarkers are measured in individual fish without derivatization in less than 5 minutes, and an automated instrument is adapted to process samples at the microscale level.
Aim 1 activities improve the quantification of steroid hormones in plasma to identify endocrine disrupting chemicals that elicit a physiological change in exposed fish. The steroids are recovered from low volumes of plasma, concentrated in a capillary using pH-mediated stacking, separated, and quantified.
Aim 2 translates a manual method of sample processing into an automated microscale extraction. Commercially available magnetic beads are integrated in a fused silica capillary and serve as frits that hold chromatographic packing utilized for solid phase extraction. The sophisticated robotics in the capillary electrophoresis instrument are leveraged to process single samples in minutes. The instrument decreases the working volume needed for sample handling from milliliters to microliters. When both aims are used in tandem, the method rapidly and simultaneously monitors multiple steroids in each sample. The new technology is compatible with the microliter plasma volumes associated with zebrafish. These advances make measurements of multiple steroids feasible in individual fish by reducing the required time and labor to process and analyze samples.
This proposal addresses the compelling need for new tools to study the relationship between human health and environmental endocrine disrupting chemicals. These contaminants interfere with the function of hormonal systems, leading to reproductive problems, impaired cognitive development, obesity, and cancer. New technology will be established to rapidly detect endocrine disruption using blood samples that are processed on the microscale to reduce the time required for sample handling and analysis from hours to minutes.
| Nyakubaya, Vincent T; Durney, Brandon C; Ellington, Marriah C G et al. (2015) Quantification of circulating steroids in individual zebrafish using stacking to achieve nanomolar detection limits with capillary electrophoresis and UV-visible absorbance detection. Anal Bioanal Chem 407:6985-93 |
