Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

Abstract

Title: Mechanistic Modeling of Bystander Effects: An Integrated Theoretical and Experimental Approach

Authors: Aloke Chatterjee and William R. Holley

Institutions: Life Sciences Division Lawrence Berkeley National Laboratory

The overall goal of the project is to develop a theoretical model based on mechanisms for bystander effects. In support of the modeling effort, experiments, conducted at three institutions, have already produced important information on mechanisms. For example, Les Braby and John Ford, at Texas A&M University, have data suggesting that DNA PK negative cells (AGO 1522) experience a higher rate of bystander DNA damage involving double strand breaks. Although the data are still being analyzed, preliminary results indicate a higher yield of micronuclei in the DNA PK negative cells compared with PK normal cells. If confirmed we have indirect evidence that double strand breaks are produced in bystander cells. Using the same cell line, AGO1522, Kathy Held of Massachusetts General Hospital has also demonstrated a substantial bystander effect in the formation of micronuclei (a four fold increase in damage) at doses as low as 10 cGy. In unirradiated bystander cells, the percentage of cells with micronuclei increase from the background level of about 2% to about 8% at all doses from 10 cGy to 5 Gy to the hit cells. Kathy Held has used 250kVp x-rays and a novel technique in which the irradiated as well as the unirradiated cells share medium, but are not in direct contact with each other. Her data indicate that the extent of the bystander effect is independent of dose in the range 10 cGy to 5 Gy. Kevin Price and Barry Michael at the Gray Laboratory have obtained similar results on micronuclei formation using soft x-rays. We are very close to reaching the conclusion that double strand breaks are produced in bystander cells. The development of our theoretical model starts with the following questions: (i) how are the double strand breaks produced?; and (ii) why do yields of these breaks saturate in the bystander cells? In order to answer these questions we make several hypotheses: (a) double strand breaks are produced by –OH type radicals formed in clusters with each cluster containing a certain number of radicals; (b) each radical in a cluster is produced through a signaling mechanism as a result of a class of cytokines binding to receptors; and (c) saturation in the yield of double strand breaks and therefore in the formation of micronuclei occurs due to the saturation of the receptors accessible to cytokines. In order to have a quantitative baseline we have made some calculations to determine the number of clusters needed to form a double strand break. In the absence of any knowledge on the exact locations of the formation of clusters within a cell nucleus, we have considered radicals produced either randomly inside the nuclear volume (5 µm radius in our model) or in close proximity to chromatin fibers. As a stating point in these calculations we have considered six –OH type radicals in each cluster. Preliminary results of these calculations indicate that, on average, one double strand break is produced for every 764 clusters of OH radicals randomly distributed within the nucleus. If we consider only OH radical clusters generated within one OH diffusion distance of the chromatin fibers in an interphase cell nucleus we find that an average of 107 clusters are required to produce one double strand break. Further calculations are being carried out, within the parameters of our strand break rejoining model, to determine the number of double strand breaks induced in bystander cells needed to reproduce the yields of micronuclei obtained in the experimental arms of this project.

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