Abstract:

These are a series of comparative responses of human aryl hydrocarbon receptor (AhR) signaling to MC252 oil fractions. The material was fractionated oil by D. Pangeni and E. Overton, collaborators at LSU using a silica gel chromatography method with hexane as the starting solvent and mixing with dichlormethane up to 50% final volume to resolve the aromatic hydrocarbons. 50 ul aliquots were dried and sent to the Miller lab for human AhR signaling and other toxicity testing. YCM3 yeast was exposed to varying dilutions of the 27 oil fractions and assessed for AhR signaling with a lacZ reporter assay. The greater the dilution that retains signing ability, the more potent and/or abundant the PAH ligand(s) are in the sample. Two strong peaks of activity are seen for fractions 17-18 and 20-24. These fractions contain different PAHs indicated in the chart at the end of the data. Compounds of interest in the fractions include methyl-substituted chrysenes.

Suggested Citation:

Charles Miller, Ahmad Alqassim, Deepa Pangeni, Edward Overton. 2017. Human aryl hydrocarbon receptor (AhR) signaling responses to MC252 oil fractions in YCM3 yeast. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N70R9MTM

Purpose:

To identify MC252 oil fractions with the highest activity for aryl hydrocarbon receptor activation.

Data Parameters and Units:

absorbance @ 600 nm (arbitrary units), absorbance @ 405 nm (arbitrary units), beta-NF = beta naphthoflavone, positive control ligand, LacZ ((absorbance at 405 nm - 0.16)*1000/(Absorbance at 600 nm * 20 minutes assay time * 0.025 mL cell suspension volume)), units = arbitrary, MC252 fraction (1-27) The lacZ units are arbitrary units from the reporter assay that loosely reflect the amount of beta-galactosidase activity due to lacZ gene expression triggered by the oil ligand's binding to AhR and subsequent activation of lacZ gene transcription.

Methods:

General methods used are described in: Fox, J.E., Burow, M.E., McLachlan, J.A., Miller, C.A. 2008. Detecting ligands and dissecting nuclear receptor-signaling pathways using recombinant strains of the yeast Saccharomyces cerevisiae. Nature Protocols. 3: 637-645. doi: 10.1038/nprot.2008.33

Instruments:

Data are generated from a Biorad plate reader performing in the visible wavelengths (600 and 405 nm).

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