The Broad Spectrum of Responses to Oxidants in Proliferating Cells: A New Paradigm for Oxidative Stress

Proliferating mammalian cells exhibit a broad spectrum of responses to oxidative stress, depending on the stress level encountered. Very low levels of hydrogen peroxide, e.g., 3 to 15 mu M, or 0.1 to 0.5 mu mol/107 cells, cause a significant mitogenic response, 25% to 45% growth stimulation. Greater concentrations of H2O2, 120 to 150 mu M, or 2 to 5 mu mol/107 cells, cause a temporary growth arrest that appears to protect cells from excess energy use and DNA damage. After 4 6 h of temporary growth arrest, many cells will exhibit up to a 40‐fold transient adaptive response in which genes for oxidant protection and damage repair are preferentially expressed. After 18 h of H2O2 adaptation (including the 4‐6h of temporary growth arrest) cells exhibit maximal protection against oxidative stress. The H2O2 originally added is metabolized within 30‐40min, and if no more is added the cells will gradually de‐adapt, so that by 36h after the initial H2O2 stimulus they have returned to their original level of H2O2 sensitivity. At H2O2 concentrations of 250 to 400 mu M, or 9 to 14 mu mol/107 cells, mammalian fibroblasts are not able to adapt but instead enter a permanently growth‐arrested state in which they appear to perform most normal cell functions but never divide again. This state of permanent growth arrest has often been confused with cell death in toxicity studies relying solely on cell proliferation assays as measures of viability. If the oxidative stress level is further increased to 0.5 to 1.0 mM H2O2, or 15 to 30 mu mol/107 cells, apoptosis results. This oxidative stressinduced apoptosis involves nuclear condensation, loss of mitochon/drial transmembrane potential, degradation/down‐regulation of mitochondrial mRNAs and rRNAs, and degradation laddering of both nuclear and mitochondrial DNA. At very high H2O2 concentrations of 5.0 to 10.0 mM, or 150 to 300 mu mol/107 cells and above, cell membranes disintegrate, proteins and nucleic acids denature, and necrosis swiftly follows. Cultured cells grown in 20% oxygen are essentially preadapted or preselected to survive under conditions of oxidative stress. If cells are instead grown in 3% oxygen, much closer to physiological cellular levels, they are more sensitive to an oxidative challenge but exhibit far less accumulated oxidant damage. This broad spectrum of cellular responses to oxidant stress, depending on the amount of oxidant applied and the concentration of oxygen in the cell culture system, provides for a new paradigm of cellular oxidative stress responses.