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 > 24 h. 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 3 h treatment of cells, which correlated with a decrease of ATP levels (R 2  > 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.