Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines.

Steroid 5-α reductase (5AR) is responsible for the reduction of steroids to 5-α reduced metabolites, such as the reduction of testosterone to 5-α dihydrotestosterone (DHT). A new adverse outcome pathway (AOP) for 5AR inhibition to reduce female reproduction in fish (AOP 289) is under development to clarify the antiestrogenic effects of 5AR inhibitors in female fish. A sensitive method for the DHT analysis using chemical derivatization and liquid chromatography-tandem mass spectrometry was developed. A cell-based 5AR inhibition assay that utilizes human cell lines, a transient overexpression system, and fish cell lines was developed. The measured IC50 values of two well-known 5AR inhibitors, finasteride and dutasteride, were comparable in the different systems. However, the IC50 of dutasteride in the fish cell lines was lower than that in the human cell lines. Finasteride showed a higher IC50 against the RTG-2 cell line. These results demonstrated that 5ARs inhibition could differ in terms of structural characteristics among species. The assay has high sensitivity and reproducibility and is suitable for the application in 5AR inhibition screening for various endocrine disruption chemicals (EDCs). Future studies will continue to evaluate the quantitative inhibition of 5AR by EDCs to compare the endocrine-disrupting pathway in different species.

Subunit δ is the key player for assembly of the H(+)-translocating unit of Escherichia coli F(O)F1 ATP synthase.

The ATP synthase (F(O)F1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γε and the stator ab2α3ß3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F(O)F1 complexes by affinity chromatography with the use of mutants synthesizing different sets of F(O)F1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F(O)F1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of F(O)F1, F1 subunit δ is the key player in generating stable F(O). Subunit δ serves as clamp between ab2 and c10α3ß3γε and guarantees that the open H(+) channel is concomitantly assembled within coupled F(O)F1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H(+)-translocating enzymes.