A FACS‐Based Reporter Assay Prototype Employing the Mouse AHR Protein Sensitively and Autonomously Detects Dioxins in Amphibian Cells

Cultured amphibian cells are well known to be highly resistant to osmotic stress and can readily be exposed to freshwater, making them an ideal sentinel system to monitor environmental contaminations. However, the fact that amphibians possess a low-activity AHR protein has precluded their use in dioxin detection assays. To circumvent this limitation, we have designed a detection prototype bestowing dioxin sensitivity to otherwise poorly responsive amphibian cells. This novel assay is based on a series of dual vectors harboring (i) an inducer cassette constitutively expressing equimolar amounts of the mouse AHR with an mCherry internal control and (ii) a reporter cassette consisting in multimerized xenobiotic response elements driving GFP expression. Measurements were performed by FACS analyses, by applying the GFP/mCherry intensity normalization ratio on a cell-by-cell basis, yielding highly reproducible results. In electroporated primary cultures of the Xenopus tropicalis frog, our assay detects diverse POPs in the range of 20-100 pM (TCDD, PeCDF, and B[k]F), 1-nM (PeCDD and PCB126), or 10 nM (dBA), representing a 400-fold improvement compared with RT-PCR assays performed on frog cells and tadpoles. Amphibian cell lines harboring this vector might readily be exposed to environmental waters, thereby expanding the repertoire of available dioxin detection toolkits.