Elimination of interfering compounds in preparation for analysis by an Ah receptor based bioassay

In general it has been assumed that bioassays are less sensitive to sample clean up than chemical analysis methods. For chemical analysis methods such as gas chromatography/mass spectrometry (HRGC/HRMS) it is necessary to submit samples to extensive and expensive clean up in order to remove compounds that will interfere with the measurement of the compounds of interest. This is especially true for biological samples where compounds such as lipids must be removed to avoid compromising the performance of the instrument. In contrast bioassays tend to be more robust in relation to biological compounds and it is not necessary to remove these compounds in sample clean up. As long as the sample extract does not contain compounds that are acutely toxic to the bioassay, the results should indicate the presence of compounds that bind to and activate the Aryl hydrocarbon receptor (AhR). Concerns about sample clean up for bioassays arise when it is considered that bioassays can respond to a wide range of different compounds. Unlike chemical analysis, which measures the concentrations of specific compounds, bioassays sum the affects of all compounds present in the sample extract that can bind to and activate the AhR. For a crude extract this may include both “classical” and “non-classical” AhR ligands as described by Denison et al.1 “Classical” Ah ligands are hydrophobic compounds that have a planar, aromatic structure similar to that of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). The “classical” AhR ligands include the polyhalogenated dibenzodioxins/dibenzofurans (PHDD/F), polyhalogenated biphenyls (PHB), polybrominated diphenyl ethers (PBDE), as well as some other polyaromatic hydrocarbons (PAH). Other compounds have been identified that can bind to and activate the AhR, but do not have the same structural characteristics as the “classical” AhR ligands. These “non-classical” AhR ligands include biological molecules such as bilirubin, tryptophan metabolites, and some members of the corticosteroid group. AhR based bioassays (AhR binding or gene expression assays) do not discriminated between these various active compounds. Therefore, it is necessary to use sample clean up as a way to isolate the compounds that are of interest. Generally, this involves the isolation of PHDD/F and PHB, The two groups of “classical” AhR ligands that are currently widely regulated. Here we report on various sample preparation methods and their efficacy at removing environmental contaminants (pesticides and PAH) as well as selected “non-classical” AhR ligands.