Computational protocol: Human Colon Microbiota Transform Polycyclic Aromatic Hydrocarbons to Estrogenic Metabolites

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Protocol publication

[…] For the bioassays, we used a modified protocol from that was based on the protocol developed by for the yeast estrogen bioassay and for the yeast Ah bioassay. Briefly, these researchers transformed Saccharomyces cerevisiae with the human Ah receptor gene and the human ER-α gene, together with expression plasmids containing responsive elements and the lacZ reporter gene (encoding the enzyme β-galactosidase). The expression of β-galactosidase is triggered by test chemicals, which upon binding to the respective receptors induce the conformational change necessary for binding of the receptor/ligand dimer to the responsive elements. This β-galactosidase activity is quantified at 540 nm by the conversion of the chromogenic substance chlorophenol red–β-d-galactopyranoside into chlorophenol red. The bioassay response is expressed as the absorbance at 540 nm divided by the optical density at 630 nm [(A540/A630)net]. Positive signals in the Ah receptor assay were typically expressed as percentage equivalence to 200 nM benzo(a)pyrene, which arbitrarily corresponded to a bioassay response of 100%. Similarly, estrogenic activity of the samples was expressed as percentage equivalence to 6.96 nM 17α-ethynyl estradiol (EE2), which elicited a 100% response in the ER bioassay (). To make sure that background signals from GI suspensions of soil or food matrices did not interfere with the detection of estrogenic signals in the bioassays, corrections were made in a set of negative control experiments by subtracting the response of a PAH-containing digest from that from a blank digest without PAHs (see Supplemental Material available online at http://ehp.niehs.nih.gov/docs/2004/7259/supplemental.pdf). The bio-assays were performed in 96-well plates in which 10 μL of the test compounds was tested and incubated with 240 μL of the genetically modified yeast (optical density, 0.25 at 610 nm). Serial dilutions of the test compounds were made in dimethyl sulfoxide, which allowed generation of dose–response curves for doses (ordinate) versus activity (abscissa). The data were fitted by a four-parametric logistic model using the Marquardt-Levenberg algorithm (Sigmaplot 4.0; SPSS Inc., Chicago, IL, USA) (). […]

Pipeline specifications

Software tools SigmaPlot, SPSS
Application Miscellaneous
Organisms Homo sapiens
Diseases Colorectal Neoplasms, Drug-Related Side Effects and Adverse Reactions
Chemicals Estrogens, Ethinyl Estradiol, p-Aminohippuric Acid, Phenanthrenes, Pyrenes