Citations : ImageXpress Micro 4

We present here methods for assessing hepatotoxicity by high content imaging and image analysis. The assays focus on the characterization of toxic effects using a variety of phenotypic endpoint readouts. Multi-parametric automated image analysis is used in the protocols to increase assay sensitivity and provide important information about possible in vitro toxicity mechanisms. iPSC-derived hepatocytes were used as a model for the hepatotoxicity assays, but the methods would also be suitable for other liver toxicity cell models. The methods contain detailed step-by-step descriptions of the cell treatment, staining, image acquisition, and image analysis.

Tris (4-hydroxyphenyl)ethane (THPE), a trisphenol compound widely used as a branching agent and raw material in plastics, adhesives, and coatings is rarely regarded with concern. However, inspection of in vitro data suggests that THPE is an antagonist of estrogen receptors (ERs). Accordingly, we aimed to evaluate the antiestrogenicity of THPE in vivo and tested its effect via oral gavage on pubertal development in female CD-1 mice. Using uterotrophic assays, we found that THPE either singly, or combined with 17β-estradiol (E2) (400 μg/kg bw/day) suppressed the uterine weights at low doses (0.1, 0.3, and 1 mg/kg bw/day) in 3-day treatment of weaning mice. When mice were treated with THPE during adolescence (for 10 days beginning on postnatal day 24), their uterine development was significantly retarded at doses of at least 0.1 mg/kg bw/day, manifest as decreased uterine weight, atrophic endometrial stromal cells and thinner columnar epithelial cells. Transcriptome analyses of uteri demonstrated that estrogen-responsive genes were significantly downregulated by THPE. Molecular docking shows that THPE fits well into the antagonist pocket of human ERα. These results indicate that THPE possesses strong antiestrogenicity in vivo and can disrupt normal female development in mice at very low dosages.

The metastatic potential of breast cancer cells has been strongly associated with overexpression of the chemokine CXCL12 and the activity of its receptor CXCR4. Lidocaine, a local anaesthetic that can be used during breast cancer excision, inhibits the growth, invasion, and migration of cancer cells. We therefore investigated, in a breast cancer cell line, whether lidocaine can modulate CXCL12-induced responses.