Abstract:

Dorado well "surrogate" oil was fractionated by silica gel chromatography. Fractions were analyzed for bioactivity in the aryl hydrocarbon receptor signaling assay essential as 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. The fractions were diluted by a factor of 10x, resulting in dilutions down to 1/10E6 that retained bioactivity. Means and standard deviation of fractions assayed in internal triplicates are shown.

Suggested Citation:

Charles Miller, Ahmad Alqassim. 2017. Human aryl hydrocarbon receptor signaling in response to Dorado oil fractions. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7CF9NGV

Purpose:

Identify fractions of oil with greatest biological activity.

Data Parameters and Units:

Absorbance at 600 nm (cell density), Absorbance at 450 nm, Lac units, Oil dilutions (1:2000, 1:20000, and 1:200000, 1:2M, 1:20M, 1:200M), Dorado oil fraction (2.1-2.28)

Methods:

These are aryl hydrocarbon receptor signaling assays conducted on fractions of Dorado (surrogate for MC252) crude oil. Assay performed in triplicate on 28 fractions 2.1 through 2.28. Each dried fraction was dissolved in 100 ul dimethylsulfoxide (a 1/2 dilution) and then 2 ul diluted into 200 ul medium + YCM3 yeast. This equals a 1:200 dilution of the fraction on the top 3 wells of the plate for each sample. Then 10-fold serial dilutions were made in the wells below, giving 1:2000, 1:20,000 and 1:200,000, 1:2M, 1:20M, 1:200M dilutions. The plate for 600 nm absorbance contains fractions 2.1, 2.2, 2.3, 2.4 dilutions 400 x to 400000000x and untreated and that the mean absorbance at 600 nm was averaged and used in all calculations for LacZ unit. Results were consistent across all fractions and dilutions at 600 nm which is why only one plate was used for the average calculations. Minimal background signal of 0.16 Abs. 405 nm was subtracted in the lacZ formula. The assay ran for 20 minutes and a volume of 0.025 ml cells was used per each point. LacZ units=(abs. 405 nm in well - background 405 nm of 0.097) *1000/absorbance 600 nm of 0.318 * 15 min assay time * 0.05 ml cells used in assay then the means and std. deviations (SD) were calculated for each triplicate condition. LacZ units are arbitrary and reflect the relative amount of beta-galactosidase enzyme activity due to transactivation of the lacZ gene by the ligand activated human AhR/Arnt protein complex. Any negative unit values were artifactual and were removed. The green cells are positive reference controls treated with B-NF @ 1 uM. In % calculations these represent 100% LacZ signaling for normalization. A B-NF dose response is at the bottom of table. Aryl hydrocarbon receptor signaling assay essential as 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:

Bio-Rad plate reader used to collect absorbance data at 405 nm (nitrophenol signal in lacZ assay) and 600 nm (cell density reading).

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