Activation of molecular oxygen to reactive forms is common in biological systems. These activated forms can cause changes in DNA structure which can lead to induction of cancer. It is believed that metals, primarily iron, are required to enhance production of such activated forms of oxygen and to convert relatively innocuous forms into more reactive species such as the hydroxyl radical. Much is being learned about the reaction properties of iron under cell-free conditions and the nature of iron-mediated hydroxyl radical damage to DNA. It is recognized that cells normally maintain strict control over intracellular metals. Yet, the cellular forms of metal which lead to DNA damage under conditions of oxidative stress remain poorly characterized. these may take protein or trace """"""""low molecular weight"""""""" forms. It also recognized that the chemical nature of such forms determines their role in cellular effects. Our overall goal is to identify the forms of intracellular iron which contribute to inhibition of cell proliferation under oxidative stress and to characterize the nature of this participation. The following specific aims are proposed: (1) to compare the efficiency of DNA strand break production in cells of differing iron speciation subjected to forms of oxidative stress including H2O2, extracellular O2-, and intracellular O2- generated by action of cellular enzymes; (2) to characterize the biochemical pathway(s) which supply iron utilized in the strand break reaction; and (3) to derive probable candidates for intracellular iron species which react to produce DNA damage. to these ends, a human cell line in which the overall iron content can be reduced as much as five- fold without apparent detriment will be used. Additional tools comprise nuclear and plasmid forms of DNA which will be reacted with specific low molecular weight and protein iron species in buffers which have been rigorously depleted of trace reactive iron. On the basis of the hypothesis that low molecular weight iron species will associate with DNA in characteristic stereochemistries, a model for cellular iron species which react to cause breakage will be derived from comparison of results of experiments with each strand breakage system.
