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 oxid ative 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/10(7) cells, c ause a significant mitogenic response, 25% to 45% growth stimulation. Great er concentrations of H2O2, 120 to 150 mu M, or 2 to 5 mu mol/10(7) cells, c ause a temporary growth arrest that appears to protect cells from excess en ergy 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 fo r oxidant protection and damage repair are preferentially expressed. After 18 h of H2O2 adaptation (including the 4-6 h of temporary growth arrest) ce lls exhibit maximal protection against oxidative stress. The H2O2 originall y added is metabolized within 30-40 min, and if no more is added the cells will gradually de-adapt, so that by 36 h after the initial H2O2 stimulus th ey have returned to their original level of H2O2 sensitivity. At H2O2 conce ntrations of 250 to 400 mu M, or 9 to 14 mu mol/10(7) cells, mammalian fibr oblasts are not able td adapt but instead enter a permanently growth-arrest ed state in which they appear to perform most normal cell functions but nev er divide again. This state of permanent growth arrest has often been confu sed with cell death in toxicity studies relying solely on cell proliferatio n 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/10(7) cells, apoptosis results. This oxidative stress-induced apoptosis involves nuclear condensa tion, loss of mitochondrial transmembrane potential, degradation/down-regul ation of mitochondrial mRNAs and rRNAs, and degradation/laddering of both n uclear and mitochondrial DNA. At very high H2O2 concentrations of 5.0 to 10 .0 mM, or 150 to 300 mu mol/10(7) cells and above, cell membranes disintegr ate, proteins and nucleic acids denature, and necrosis swiftly follows. Cul tured cells grown in 20% oxygen are essentially preadapted or preselected t o survive under conditions of oxidative stress. If cells are instead grown in 3% oxygen, much closer to physiological cellular levels, they are more s ensitive to an oxidative challenge but exhibit far less accumulated oxidant damage. This broad spectrum of cellular responses to oxidant stress, depen ding on the amount of oxidant applied and the concentration of oxygen in th e cell culture system, provides for a new paradigm of cellular oxidative st ress responses.