Services: Environmental Protection Agency (EPA) ToxCast Assays
Vala Sciences Inc specializes in the development of assays that feature "High Content Analysis" (HCA), based upon imaging of cultured cells and tissue via automated high throughput microscopy workstations and automated digital image analysis.
Vala has recently contracted with the EPA to perform a variety of novel cell based assays as part of the ToxCast program. These assays are available as part of Vala's growing contract service offering.
EPA ToxCast News Release Link: yosemite.epa.gov/opa/admpress.n…
More information on ToxCast: http://epa.gov/ncct/toxcast/
More information on ToxCast database: http://actor.epa.gov/actor/faces/ToxCastDB/Home.jsp
The EPA can be divided into the following broad categories:
1. Assays based upon lipid droplets and associated proteins.
Lipid droplets are found in hepatocytes, adipocytes, and skeletal muscle, and are relevant to a variety of pathways and disease processes, including fatty liver disease (a precursor condition to fibrosis, cirrhosis, and hepatocellular carcinoma). Lipid droplet related assays include assays for:
a. Adipogenesis. Test compounds are tested for their influence on the process of differentiation of adipocytes from precursor cells.
b. Lipid droplet content. Effects of test compounds are evaluated for their ability to influence the lipid droplet content of hepatocytes or adipocytes (to test for potential anti-obesity, or pro-obesity effects).
c. Lipid droplet formation. Effects of test compounds on lipid droplet formation induced by exposure to fatty acids.
d. Lipid droplet-associated proteins. Effects of test compounds on the expression of lipid droplet associated proteins (e.g., perilipin, which is specific for adipocytes, or ADFP, which is found in both adipocytes and hepatocytes) are evalutated.
e. Lipolysis. Effects of test compounds on phosphorylation of hormone sensitive lipase (HSL) and/or perilipin, and the colocalization of these proteins with lipid droplets; phosphorylation and colocalization of perilipin and HSL with lipid droplets is a key step in the initiation of lipolysis.
2. Assays based upon quantifying membrane proteins. Many biological phenomena involve translocation of proteins to or from the plasma membrane, or changes in the expression of proteins at the plasma membrane; membrane associated proteins are also involved in cell to cell adherence, which is a key mechanism in embryonic differentiation and tumor formation and metastasis. Vala’s assays based upon membrane proteins include:
a. Beta-catenin. The assay measures the degree of association of beta-catenin with the plasma membrane, cytoplasm, and nuclear compartments. Beta-catenin typically relocates to the nucleus as a result of activation of the Wnt pathway, which is relevant to differentiation and tumor genesis.
b. Protein kinase C. The assay measures the degree of association of protein kinase C with the plasma membrane.
c. N-cadherin. Cadherin proteins are involved in adhesion of cells to each other within tissue. Development of tumors typically involves the “epithelial to mesenchyme transition” (EMT). Epithelial cells normally express E-cadherin. During EMT, expression of E-cadherin is reduced and N-cadherin is increased. Vala’s assay for N-cadherin can be used to identity positive or negative effects of compounds on N-cadherin expression in HeLa cells.
d. E-cadherin. Vala’s assay for E-cadherin can be used to identify the effects of compounds on E-cadherin expression in A431 cells.
e. Pan-cadherin. Vala’s assay for “pan-cadherins” detects and quantifies the overall expression of classic cadherin proteins. Cadherin proteins, in general, are typically downregulated in tumor cells. The pan-cadherin assay can thus be used to test for potential effects of compounds on the overall expression level of cadherins, which is an index of their metastatic potential of the cell (the less cadherins are expressed, the more easily tumor cells can migrate through tissues).
3. Assays based upon analyzing nuclear count, DNA content, or nuclear protein expression. Nuclear assays offered by Vala include:
a. Cell count. For assays utilizing DAPI or Hoechst staining, all nuclei are counted, giving an estimate of cell count. This is often multiplexed with other assays.
b. DNA content. For all of the HCA assays offered by Vala, the DNA content of the cells is quantified, leading to the production of ploidy histograms.
c. Histone acetylation. Vala’s histone acetylation assay quantifies the effect of test compounds on nuclear acetyl-H3, which is a critical regulator of chromatin structure.
4. Differentiation and function of cells derived from stem cell cultures. Vala, in collaboration with the Sanford Burnham Medical Research Institute, has developed methods and instrumentation to quantify electrically stimulated calcium transients in cardiac myocytes emerging from cultures of either embryonic or induced pluripotent stem cells. Assays include:
a. Analysis of compounds on the ability of electrical stimuli to elicit calcium transients in differentiating cardiac myocytes. For stem cell cultures plated in 96-well dishes, the cells are loaded with an intracellular calcium indicator and Hoechst (to provide a nuclear stain); the instrument then automatically positions an electrode assembly into each well, delivers a series of electrical stimulation, and records digital video clips of the fluorescence which is proportional to intracellular calcium. Vala’s CyteSeer program then analyzes the calcium transients for each cell imaged per well. Compounds are tested for their ability to inhibit or potentiate calcium transient formation, and for their effects on calcium kinetics. Compounds are also evaluated for their ability to induce arrhythmias. Cells can be fixed and labeled for biomarkers following the kinetic experiments, for the identified cells, providing additional characterization of the cells. The nuclear Hoechst signal can also be evaluated for effects on ploidy or apoptosis.
5. Mitochondrial expression/function.
a. Mitochondrial membrane potential. Vala’s assay can quantify the effects of test chemicals on the mitochondrial membrane potential, which is critically important in mitochondrial function in oxidative metabolism, and the process of apoptosis.
