title: Hepatic co-cultures in vitro reveal suitable to detect Nrf2-mediated oxidative stress responses on the bladder carcinogen o-anisidine.
abstract: The azo dye o-anisidine is known as an industrial and environmental pollutant. Metabolites of o-anisidine remain in the liver for >24h. However, the toxicological impact of o-anisidine on the liver and its individual cell types, e.g., hepatocytes and immune cells, is currently poorly understood. A novel co-culture system, composed of HepG2 or Huh-7 cells, and differentiated THP-1 cells was used to study the metabolic capacity towards o-anisidine, and compared to primary murine hepatocytes which express high enzyme activities. As model compounds the carcinogenic arylamine o-anisidine and its non-carcinogenic isomer, p-anisidine, as well as caffeine were used. Global proteome analysis revealed an activation of eIF2 and Nrf2-mediated oxidative stress response pathways only in co-cultures after treatment with o-anisidine. This was confirmed via detection of reactive oxygen species. In addition, the mitochondrial membrane potential decreased already after 3h treatment of cells, which correlated with a decrease of ATP levels (R2>0.92). In the supernatant of co-cultured, but not single-cultured HepG2 and Huh-7 cells, o-anisidine caused increases of damage-associated proteins, such as HMGB1 (high mobility group box-1) protein. In summary, only co-cultures of HepG2 and THP-1 cells predict o-anisidine induced stress responsive pathways, since the system has a higher sensitivity compared to single cultured cells.
authors.affiliation: Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany. Electronic address: franziska.wewering@bfr.bund.de.
authors.forename: Franziska
authors.initials: F
authors.lastname: Wewering
authors.affiliation: Department of Molecular Systems Biology, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Florent
authors.initials: F
authors.lastname: Jouy
authors.affiliation: Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.
authors.forename: Sükran
authors.initials: S
authors.lastname: Caliskan
authors.affiliation: Department of Molecular Systems Biology, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany; Department of Bioanalytics, University of Applied Sciences and Arts of Coburg, 96450 Coburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kalkhof
authors.affiliation: Department of Molecular Systems Biology, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany; Department of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany; Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.affiliation: Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Luch
authors.affiliation: Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Zellmer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000814:Aniline Compounds / Q000633:toxicity*; D002110:Caffeine / Q000633:toxicity; D002273:Carcinogens / Q000633:toxicity*; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D002273:Carcinogens; D016207:Cytokines; D051267:NF-E2-Related Factor 2; D017382:Reactive Oxygen Species; D002110:Caffeine; C013813:4-anisidine; D000255:Adenosine Triphosphate; C003898:2-anisidine
keywords: Co-culture; HepG2; Nuclear factor erythroid 2-related factor 2 (Nrf2); ROS; o-Anisidine