Impact of culture conditions on oxidative stress responses in HepG2 cells

Reliable, mechanism-based in vitro assays are essential for Next Generation Risk Assessment of non-genotoxic carcinogens, including those operating through the induction of oxidative stress. This study evaluated how oxygen tension, metabolic capacity and exposure scenarios influence oxidative stress quantification in human hepatocytes, aiming to support the development of in vitro assays relevant for chronic toxicity. We compared chemically-induced oxidative stress in HepG2 cells under atmospheric (20% O₂) and physiological (3% O₂) conditions by measuring reactive oxygen species (ROS) and the ratio between reduced and oxidized glutathione (GSH/GSSG). Lower oxygen tension reduced cell clustering, slowed proliferation, and mildly decreased hepatocyte marker expression and cytochrome P450 activity. Notably, the potency to induce ROS of most chemicals was lower at 3% oxygen, while the potency to perturb GSH/GSSG was chemical dependent. Maturation of HepG2 cells (mHepG2) yielded a more hepatocyte-like phenotype, with increased expression of metabolic genes and cytochrome P450 activity. Six of the eight tested chemicals showed greater susceptibility to chemically-induced ROS in mHepG2 cells. However, mHepG2 cells had a seemingly better recovery capacity based on the chemical's potencies to perturb GSH/GSSG. Importantly, mHepG2 cells can be cultured over extended periods, allowing to study the effects of repeated exposure. Repeated exposure with or without recovery periods resulted in increased potency to induce oxidative stress. These findings highlight the importance of oxygen tension, metabolic competence, and exposure frequency for hazard characterization of oxidative stress-inducing chemicals, supporting the development of more predictive in vitro assays for chronic toxicity.