Depletion of cellular glutathione by conditions used for the passaging of adherent cultured cells

Cultured cells are commonly exposed to trypsin-containing solutions in order to prepare cell suspensions suitable for subculture. Conditions used to release and disperse monolayers of cultured murine hepatoma 1c1c7 and human breast epithelial MCF10A cells caused the loss (40-95%) of cellular glutathione (GSH), but did not affect viability. Glutathione contents returned to pretrypsinization values within 24 h of replating. In contrast, the GSH contents of cultured rat hepatoma 5L cells were not affected by trypsinization. Exposure of 1c1c7 cultures to H2O2 or etoposide 1 or 24 h after replating resulted in concentration-dependent cytostatic and cytotoxic effects. The concentration-response curves defining the cytostatic and cytotoxic effects of etoposide, and the cytostatic effects of H2O2 were not influenced by the timing of toxicant addition. However, 1c1c7 cultures treated with H2O2 1 h after replating were more susceptible to the cytotoxic actions of the peroxide than cultures treated 24 h after plating. These studies show that conditions commonly used for the passaging of cultured cells can lead to a transient, but profound loss of GSH in some cell lines, Furthermore, the outcome of cytotoxicity analyses call be influenced by the time elapsed between the plating of cultures and the addition of toxicant. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.