Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

Abstract

Authors: LM Green, BM Bianski, E Kyeyonne-Nyombi, L Ortloff & Pinal Pandya
Radiobiology Program, Chan Shun Pavilion room A1010Loma Linda, CA 92354
E-mail lgreen@dominion.llumc.edu

INTRODUCTION:
The thyroid, like most epithelial glands are a common site of tumor development and sensitive to ionizing radiation induced carcinogenic changes. We have established thyroid tissue analogs in vitro that better approximates the in vivo situation. The thyroid tissue analogs produced under micro gravitational forces dramatically affects differentiation and growth patterns facilitating threedimensional assembly of the thyrocytes into mini thyroid glands. Cells grown in the low-shear stress rotating-wall vessels allow us to evaluate various aspects of complex interactions that are not possible using traditional culture techniques. For example, bioreactor technology allows for monitoring of assembled thyroid analogs exposed to chronic radiation with continuous sampling of the tissue.

Thus, our well characterized model provided us the opportunity to investigate how a relatively normal tissue responds to ionizing radiation at multiple levels, including: cell-cell exchanges (bystander), signal transduction, functional changes and modulation of gene expression. We had significant preliminary data on structural, functional, signal transduction and gene expression from acute exposures at high doses and rates (50-100 cGy, at 1 Gy/minute) of gamma, protons and iron ions. The goal of the currently funded project was to characterize the pattern of radiation modulated gene expression in thyroid tissue functional units (FTU’s) using low-dose/low-dose rate ionizing radiation.

We hypothesized that-- a low-dose/low-dose rate “priming” exposure to radiation would invoke a suite of cell and tissue level homeostatic mechanisms that should be reflected by alteration of their expressed gene products and complementary structural and functional changes in component cells and their functional tissue units (FTU’s, i.e. follicles). Once the thyrocytes complete the “transition” to the “primed” state, the thyroid FTU’s should exhibit an altered response to an acute challenge
exposure to radiation that increases their resistance to cytopathological changes relative to “unprimed” cells.

The defined specific aims are --Aim 1, thyroid tissue analogs will be irradiated with acute single doses (0, 10, 20, 50 and 200 rads) of gamma radiation with harvest times at 3 hours, 2 and 7 days post-irradiation. Protracted exposures of the same dose range and harvest points will be delivered over 2 and 7 days. Aim 2, is to challenge the low dose irradiated tissue with a subsequent exposure. The challenge doses will be equivalent to the priming dose, and in additional experiments the tissue will be exposed to 100rad 250MeV protons. The last phase (Aim 3) determines out of field effects (bystander) to measure the extent to which low dose-induced signals, identified in Aims 1 and 2, are transferred to or expressed in neighboring cells within the contiguous follicle and or adjacent follicular thyrocytes. These experiments will be accomplished by several different methods, including use of a microcollimated 70MeV proton beam and delivery of focused soft X-rays (Al-K) to individual thyrocytes within follicles using a microbeam in conjunction with our co-investigator at the Gray
Laboratory (Dr. Kevin Prise).

Currently, we have completed the comparison between flask and bioreactor grown
cells/tissues, and acquired samples of tissue and RNA from the acute low dose schedule, in duplicate. The microarrays for the acute low dose samples are being analyzed. We have obtained the source (Co-57) for the chronic low-dose/low-dose rate exposures which should commence shortly. This presentation details our findings, thus far, including the results of the 2-dimensional versus threedimensionally grown thyroid follicular cultures and preliminary differential gene products expressed in response to acute exposure to low dose gamma radiation.

RESULTS:
Differences between 2-dimensional and 3-dimensional tissue assemblies-- The thyroid cells grown in flasks assemble into follicles that remain as one or two follicular assemblies, whereas in the bioreactors the follicles form multifollicular assemblies. The bioreactor grown thyroid tissue analogs are more representative of a tissue and therefore should provide a better model for radiation effects. We have compared the gene products expressed in flasks versus bioreactor tissue exposed
to gamma radiation (0, 50 and 200 cGy). The Affymetrix gene chip contains all characterized rat genes (15, 916). The flask grown cells express 7339 genes and the bioreactor cells express 8229, with from 150 to 500 gene products increased or decreased under the various parameters. The number of increased and decreased gene products in common was surprisingly low (3-10%). This suggests there are unique differences in the 2-dimension compared to the 3-dimensionally derived
tissue sources. The fact that the tissue analogs expressed more total genes than the flask grown cells suggests that the FTU’s are biologically more complex than the 2-dimensional cultures.

Temporal changes in bioreactor generated FTU gene products following acute low-dose exposures-- Gene product profiles from the baseline, acute low-dose exposures are now being analyzed, selected products, most of which have been confirmed at the protein level are shown in Table 1.

Bioreactor Acute Single Exposure to Low Dose Gamma Rays

The most obvious feature is the shift in expression at 7days compared to the 3 hour and 2 day samples. Two interesting proteins were found altered under these conditions. The first is the increase in “proliferating cell nuclear antigen” (PCNA) which is decreased at 3 hours but increased at 7 days. The sustained late increase in PCNA may indicate that the surviving cells are increasing their proliferative status. The protein expression of PCNA confirms the levels detected by microarray
analysis. We also confirmed the reciprocal pattern for superoxide dismutase-3 (SOD3, extracellular form of SOD 1), which was increased at 3 hours but markedly decreased at 7 days.

CONCLUSIONS:

The data analysis is preliminary at this phase of the study; however, very interesting patterns are emerging from the acute exposure data. There are significant changes occurring at 7 days that we would not have predicted. The definitive assessment of our proposed hypothesis awaits the completion of the other phases of our project, namely the chronic “priming” plus/minus acute challenge exposures. Ultimately, our results will translate to risk assessment, potentially re-defining limits for radiation workers, enhancing radiotherapy by pre-dosing, and contributing to our understanding of the consequences of chronic exposure during long duration space flight.

ACKNOWLEDGMENTS: This work supported by DOE grant #DE-FG03-02ER63448 (PI, LM Green)