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Toxicol In Vitro. 2017 Apr;40:153-160. doi: 10.1016/j.tiv.2017.01.006. Epub 2017 Jan 12.

Hepatic co-cultures in vitro reveal suitable to detect Nrf2-mediated oxidative stress responses on the bladder carcinogen o-anisidine.

Author information

1
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.
2
Department of Molecular Systems Biology, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany.
3
Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.
4
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.
5
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, BrĂ¼derstrasse 34, 04103 Leipzig, Germany; Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg, Denmark.

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.

KEYWORDS:

Co-culture; HepG2; Nuclear factor erythroid 2-related factor 2 (Nrf2); ROS; o-Anisidine

PMID:
28089782
DOI:
10.1016/j.tiv.2017.01.006
[Indexed for MEDLINE]

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