科研之友
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摘要
Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) alpha or beta isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered beta-galactosidase enzyme (beta-gal) for the binding to ER alpha or beta, thus quantitatively altering the formation of enzymatically active beta-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ER alpha or beta concentrations, it was found that luminescent signals were sustainably emitted for 9 h, and 40 nM ER alpha or beta in the system would lead to the most sensitive luminescence response. Using 17 beta-estrodiol (E-2) and genistein as the representative estrogenic hormones, their binding affinities for ER alpha and beta were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on beta-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ER alpha or beta isoforms. The binding affinities of PFIs for ER alpha were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks.
