SBIR-STTR Award

Exposure to specific polychlorinated diaromatic hydrocarbons (PCDH), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, produces a wide variety of toxic/biological effects, many of which are mediated by the Ah receptor (AhR), a ligand (PCDH)-dependent transcription factor which activates gene expression. The high correlation between the ability of PCDHs to bind to the AhR and induce gene expression has allowed development of a mechanistically-based recombinant cell bioassay system which rapidly responds to PCDHs with high levels of induction of firefly luciferase. This recombinant cell line can be used as a bioassay for the rapid detection and relative quantitation of PCDHs and related chemicals present in biological and environmental samples. To develop a marketable and fully validated assay system using this cell line, we will optimize and standardize the assay in a 96 well microplate format, which will allow assay automation and the highest possible sample throughput. Sample microextraction/purification procedures for PCDHs, based on established techniques, will also be adapted for use in this new bioassay, which can tolerate lower sample purity than currently used GC-MS systems. Validation of the bioassay system will be carried out using serum previously assayed for dioxins and PCDHs.Proposed commercial application:(Not available)National Institute of Health Sciences (NIEHS)

Exposure to polychlorinated diaromatic hydrocarbons (PCDHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin produce a wide variety of toxic and biological effects. Many of these effects are mediated by the Ah receptor (AhR), a ligand (PCDH)-dependent transcription factor which activates gene expression. The chemical specific and high affinity nature of the AhR-dependent mechanism has allowed development of a recombinant mammalian cell bioassay system (the Chemical Activated Luciferase Expression (CALUX) assay) which responds to PCDHs with the induction of firefly luciferase. Initial development, optimization and validation of the CALUX microplate bioassay system and development of selective sample preparation techniques for separation of various classes of PCDHs was carried out during Phase I. We propose to complete the optimization and validation of the CALUX bioassay system as well as several microextraction procedures for PCDHs. We will also develop a validated CALUX microplate bioassay kit which can be marketed to researchers, regulators and/or industry for use in PCDH detection. The analytical and bioanalytical techniques developed here will provide a rapid and inexpensive, high sample throughput screening bioassay for the detection and relative quantitation of PCDHs and related chemicals in extracts of biological, environmental and industrial samples. PROPOSED COMMERCIAL APPLICATIONS: 2,3,7,8-tetrachlorodibenzo-p-dioxin has recently been defined by the National Toxicology Program as a human carcinogen. Once validated, the CALUX assay will provide an inexpensive method of screening for dioxins and similar chemicals in biological, environmental and industrial samples. In addition to the advantages of being faster and less expensive, smaller sample sizes can be analyzed by the CALUX assay than with current analytical methods. All of these advantages should make the CALUX assay competitive after development and validation has been completed.