title: Modulation of benzo[a]pyrene-induced p53 DNA activity by acrolein.
abstract: Acrolein, a highly electrophilic alpha,beta-unsaturated aldehyde, is by far the most reactive amongst the aldehydes present in smoke. The relative contribution of acrolein to complex mixture toxicity of smoke at the molecular level remains unknown. The current study examines the ability of acrolein to modulate the effect of benzo[a]pyrene (B[a]P), a major carcinogen found in smoke, on p53. Exposure of human lung adenocarcinoma A549 cells to 1 mM B[a]P for 48 h strongly activated the expression of p53 as seen by western blotting, and its DNA binding as shown by an electrophoretic mobility shift assay. Treatment of A549 cells with a non-lethal dose of acrolein alone (50 fmol/cell for 0.5 h) depleted 80% of total cellular glutathione but had no effect on basal p53 protein levels. When B[a]P-treated cells (48 h) were exposed to acrolein for 0.5 h there was also no effect on B[a]P-induced p53 protein levels. However, acrolein treatments profoundly inhibited the DNA binding of p53 under both basal and B[a]P-induced conditions. Depleting glutathione with buthionine sulfoximine in B[a]P-treated cells to levels similar to those obtained with acrolein decreased p53 DNA binding substantially less than with acrolein. Using a p53 dual luciferase reporter assay, acrolein caused an 83% decrease in the p53 activity induced by B[a]P (1 mM for 24 h post-transfection). The p53 protein that was immunoprecipitated after acrolein treatment was reactive with an anti-acrolein antibody indicating covalent modification. Results from this study suggest that acrolein can inhibit p53 DNA binding and activity by direct covalent modification as well as alteration of intracellular redox status. As both acrolein and B[a]P are found in cigarette smoke, this type of interaction may play an important role in the initiation of lung cancer by altering the tumor suppressor activity of p53.
authors.affiliation: Division of Toxicological Sciences, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA. sbiswal@jhsph.edu
authors.forename: Shyam
authors.initials: S
authors.lastname: Biswal
authors.forename: Terrie
authors.initials: T
authors.lastname: Maxwell
authors.forename: Tirumalai
authors.initials: T
authors.lastname: Rangasamy
authors.forename: James P
authors.initials: JP
authors.lastname: Kehrer
mesh_terms: D000171:Acrolein / Q000494:pharmacology*; D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53; D001564:Benzo(a)pyrene; D019328:Buthionine Sulfoximine; D000171:Acrolein; D008156:Luciferases; D005978:Glutathione