Analysis of Steroidogenesis Using SEP and DEST Assays
The H295R Steroidogenesis Assay is a crucial in vitro experimental method, codified under the US Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP) Test Guideline 890.1550. This assay is designed to evaluate whether a chemical affects steroid hormone production by the adrenal human H295R cell line, a well-established steroidogenic model.
Key Steps in the H295R Steroidogenesis Assay
The assay consists of six primary steps, carried out within the framework of the Standard Evaluation Procedure (SEP) and Data Entry Spreadsheet Table (DEST).
- Cell Culture Preparation
- Human adrenal carcinoma H295R cells are grown and maintained under standardized conditions before being cultured to a defined confluence (typically ~80%) in appropriate medium.
- Chemical Exposure
- Cells are treated with test substances at multiple concentrations, including controls like forskolin for stimulated steroidogenesis.
- The treatment period is usually 48 hours, allowing for steroidogenesis modulation.
- Steroid Measurement
- After treatment, culture media or cell lysate is collected for quantitative analysis of multiple steroid hormones.
- Common steroids quantified include cortisol, aldosterone, testosterone, estradiol, and progesterone.
- Analysis methods include immunoassays, ELISA, or chromatographic techniques like LC-MS/MS.
- Data Entry Using DEST
- All raw data, hormone concentrations, standard curves, controls, replicates, and exposure concentrations are recorded in the DEST spreadsheet.
- DEST standardizes data capture for computational analysis and evaluation.
- Evaluation by Standard Evaluation Procedure (SEP)
- The SEP is a systematic approach for analysing DEST data to determine the steroidogenic activity of the test chemical.
- It includes reviewing assay validity, control performance, dose-response assessment, and statistical tests for hormone level changes.
- Reporting
- Qualitative and quantitative assay results are summarised, including dose-response patterns.
- Any steroid modulation consistent with receptor-mediated or enzyme-mediated interference in steroid biosynthesis is reported.
Additional Notes
- The EPA EDSP guideline 890.1550 details Standard Operating Procedure (SOP) elements to ensure reproducibility and regulatory acceptance.
- Forskolin is commonly used to stimulate the cells and reveal compound effects under stimulated steroidogenesis conditions.
- Cell viability and cytotoxicity assessments are integral to verify that steroid changes are not due to cell death.
- Data are interpreted carefully to distinguish direct steroidogenic pathways perturbation versus non-specific toxicity.
In essence, the H295R assay with SEP and DEST involves culturing human H295R cells, exposing them to test chemicals, measuring steroid output, entering structured data into DEST spreadsheets, and evaluating results using SEP criteria to assess endocrine activity of chemicals.
While the exact stepwise protocol documents (e.g., volumes, incubation times, specific spreadsheet templates) are not included explicitly in the above search results, these elements are standardized in the EPA EDSP Test Guideline 890.1550 Steroidogenesis test and associated guidance documents. For detailed SOP documents, referring directly to the EPA EDSP official guidance and supporting materials is recommended.
- The H295R Steroidogenesis Assay, a crucial method in evaluating chemical effects on steroid hormone production, can reveal potential impacts on medical-conditions such as cancer, health-and-wellness, and other medical-conditions that are affected by hormonal imbalances.
- By measuring steroid hormones like cortisol, aldosterone, testosterone, estradiol, and progesterone in the H295R Steroidogenesis Assay, researchers can gain insights into the science behind hormonal regulation and its role in medical-conditions, such as cancer and health-and-wellness.
