Abstract:

In this experiment, an engineered Saccharomyces cerevisiae (YCM3) strain was used to detect ligand induced AhR signaling in order to compare the potencies of six mono-methylchrysenes to chrysene. Data are summarized (N = 3 experiments) and expressed as effective concentrations giving 50% response (EC50). Benzo[a]pyrene was added as a second reference PAH compound for purposes of comparison. Later, the AhR signaling response of chrysene was set to a Relative Potency Factor (RPF) of 1. Compounds with a potency greater than 1 were more effective as AhR ligands, and those <1 were weaker. The statistical significance of mean differences between chrysene and other compounds were determined by ANOVA test. These data were collected by December 2017 at Dr. Miller's Laboratory located at 1440 Canal Street, Suite 2110, New Orleans, LA 70112. This dataset supports the publication: Alqassim, A. Y., Wilson, M. J., Wickliffe, J. K., Pangeni, D., Overton, E. B., & Miller, C. A. (2019). Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono‐methylchrysenes. Environmental Toxicology, 34(9), 992–1000. doi:10.1002/tox.22770

Suggested Citation:

Ahmad Alqassim, Charles Miller. 2019. The effective concentrations giving 50% response (EC50) for the chrysenes tested in the yeast AhR signaling assay. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-570w-4417

Publications:

Alqassim, A. Y., Wilson, M. J., Wickliffe, J. K., Pangeni, D., Overton, E. B., & Miller, C. A. (2019). Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono‐methylchrysenes. Environmental Toxicology, 34(9), 992–1000. doi:10.1002/tox.22770

Purpose:

An engineered Saccharomyces cerevisiae (YCM3) strain was used to detect ligand induced AhR signaling.

Data Parameters and Units:

EC50 (effective concentrations giving 50% response) Values; Chemical (the type of chemical represented by a number; 0-7); Dose (nM); and ligand-induced AHR signalling (LacZ units). Labels for the chemicals are as follow: 0="Chrysene" 1="1-Methylchrysene" 2="2-Methylchrysene" 3="3-Methylchrysene" 4="4-Methylchrysene" 5="5-Methylchrysene" 6="6-Methylchrysene" 7="Benzo[a]pyrene"

Methods:

Ligand-induced AhR signaling was detected by an engineered Saccharomyces cerevisiae (YCM3) strain. This strain was made in Dr. Miller’s laboratory (Miller, 1999). Yeast cells were genetically engineered to express the human AhR and Arnt proteins, which are components of the aryl hydrocarbon receptor complex (AHRC) that functions as a transcription factor. AHRC directs gene expression that leads to subsequent toxicity in mammalian cells, but the yeast has only the lacZ reporter gene that serves as a biomarker for the presence and potency of AhR ligands. The presence and potency of ligands was reported by the engineered YCM3 strain containing an AHRC responsive lacZ gene by expressing the β- galactosidase enzyme. The amount of β- galactosidase expressed was determined by a yellow product (nitrophenol). Cultures were exposed in triplicate to a wide range of concentrations. The lacZ assays and cell concentrations were assessed on a plate spectrophotometer at 405 nm and 600 nm respectively. Yeast were grown in synthetic glucose medium supplemented with adenine, histidine, leucine and uracil. The overnight culture (above) was diluted 1:100 into galactose medium (same medium as above but glucose is replaced with galactose as the expression inducing sugar) and dispensed into 96 well plates (200 μl/ well). The cultures were treated with chemicals (e.g. chrysene) dissolved in dimethylsulfoxide and incubated for 18 hr at 30 degrees C and then assayed. Cells were in mid-exponential growth phase when takes for analysis of reporter gene activation. (optical density at 600 nm ~ 0.5). AhR signaling background was defined by solvent (dimethylsulfoxide) signaling alone. Maximal (positive control) signaling was defined by the reporter gene response to 2 μM β-naphthoflavone (β-NF). This maximal β-NF signal was normalized to equal a 100% response. We used a Bio-Rad BenchMark Plus plate reader to scan the 96 well plates at 405 and 600 nm wavelengths. Data were imported into a Microsoft Excel spreadsheet on an Apple computer for calculation of % signaling of experimental treatments.

Provenance and Historical References:

Miller, C. A. (1999). A Human Aryl Hydrocarbon Receptor Signaling Pathway Constructed in Yeast Displays Additive Responses to Ligand Mixtures. Toxicology and Applied Pharmacology, 160(3), 297–303. doi:10.1006/taap.1999.8769

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