Methacrolein

Aryl-hydrocarbon receptor activity was measured using a stably transfected gene reporter human cell line AZ-AhR30 (link). Briefly, 2 × 10⁴ cells/well were seeded in 96-well plates and incubated for 18 hours at 37 °C and 5% CO_2, before cells were stimulated 18 h in triplets with acrolein, cinnamaldehyde, crotonaldehyde, propanal and methacrolein (all from Sigma) in increasing concentrations (0.018/0.18/1.8/9/18/45/90/180 μM) was used as negative control, whereas indirubin (0.03 μg/ml, Sigma) was used as a positive control and as AhR-antagonists resveratrol 100 μM (Sigma) and 3′-methoxy-4′-nitroflavone 5 μM (Sigma). After adding lysis buffer and a single freeze-thaw cycle, 20 μl/well of lysates were transferred into a black 96-well flat-bottom plate (Thermo Scientific) and bioluminescent reaction were started with addition of 100 μl/well of luciferase assay reagent (Promega). Chemiluminscence was measured (10 sec/well) using a spectrophotometer Tecan InfiniteM200 PRO.

Glycerol and propylene glycol, 2,4-dinitrophenylhydrazine (DNPH; 4% in phosphoric acid solution), o-phenylenediamine (o-PD), 2,4-dinitrophenylhydrazones of aldehyde/ketone–DNPH stock standard-13 (acetaldehyde–DNPH, acetone–DNPH, acrolein–DNPH, benzaldehyde–DNPH, 2-butanone–DNPH, n-butyraldehyde–DNPH, crotonaldehyde–DNPH, formaldehyde–DNPH, hexaldehyde–DNPH, methacrolein–DNPH, propionaldehyde–DNPH, m-tolualdehyde–DNPH, valeraldehyde–DNPH), methylglyoxal (40% aqueous solution), glyoxal (40% aqueous solution) and pure polyphenol standard, gallic acid, hydroxytyrosol and epigallocatechin gallate were obtained from Sigma-Aldrich (St. Louis, MO). Trizma base (Tris-(hydroxymethyl)-aminomethane ACS Reagent Grade), solvents for chromatography analysis, such as acetonitrile, methanol and water, (liquid chromatography-mass spectrometry, LC-MS grade), as well as acetic acid were purchased from Fisher Scientific (Loughborough, UK). Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Merck (Darmstadt, Germany). Fibrous 4 μm silica (“silica wool”) was obtained from H. Baumbach & Co Ltd, (Suffolk, UK) and Whatman 47 mm QMA silica filters were obtained from Sigma-Aldrich.