Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop III

Abstract

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Title: Effects of Low Dose Ionizing Radiation on Gene Expression in Human Skin.

Authors: Zelanna Goldberg(1), Chad W. Schwietert(1), Robin L. Stern (1), Michelle Arnold (2), Christine L. Hartmann Siantar (2), Robert Cary (3), Marie-Anne Descalle (2), and Bruce E. Lehnert (3).

Institutions: 1 University of California, Davis, Dept. of Radiation Oncology,
2 Laurence Livermore National Laboratory,
3 Los Alamos National Laboratory.

Significant biological effects can occur in animals, animal cells, immortalized human cell lines, and primary human cells after exposure to doses of ionizing radiation (IR) in the <1-10 cGy region. How these and other observations mimic or even pertain to the actual condition, especially in humans is unclear, though such knowledge is ultimately required for reducing the uncertainty of assessing human risks due to low dose IR (LDIR) exposures. Thus, human translational data must be obtained with which to correlate in vitro experimental findings and evaluate their “real-life” applicability. Our project uses human skin, irradiated In vivo during therapeutic radiation as a model system. Preliminary studies have focused on verifying the accuracy of the dosimetry in the low dose, out of field areas, optimizing RNA and protein extraction from the samples, assessing RNA amplification strategies and performing microarray analyses to ensure the robustness of the physics and biology components of the project prior to obtaining patient samples.

We combined measurements and PEREGRINE 3D Monte Carlo simulations to establish an overall 10-15% uncertainty predictive capability for the 18 MV clinical radiation beam used in these experiments. Based on our findings, we have altered our paradigm for sample collection: we will collect samples at the exit surface of the patient in order to optimize dosimetric accuracy and minimize dose gradient through the sample. Combining real-time measurement and exit-surface collection we anticipate overall dosimetric uncertainties of 5%.

Preliminary biologic studies have focused on obtaining global gene expression data from small volume human skin samples. Samples have been obtained from resected tissue from elective surgical procedures. 3 mm diameter core skin biopsies have been performed and samples from different areas of the body have been compared within a given person to examine homogeneity of the skin site sampled. Tissue samples are incubated up to 24 hours to assess stability of message, or they are subjected to immediate ex vivo IR at 1, 10 or 100 cGy and then incubated for equivalent times. RNA is extracted, processed, and hybridized to cDNA microarrays containing over 12,500 unique sequence validated human cDNA clones to assess gene expression changes in the samples. Expression profiles generated from amplified and unamplified RNA are being compared to confirm the fidelity of amplification schemes that are required for samples containing limited RNA.

Preliminary gene expression microarray hybridization data have suggested that as many as 116 genes have altered expression of at least a 2-fold extent following 1 cGy exposure ex vivo. These include genes that have been shown to radioresponsive in pure in vitro cell systems.