Purpose: The effect of low doses of low^linear energy transfer (photon) ionizing radiation (LDIR, <10 cGy) on human tissue when exposure is under normal physiologic conditions is of significant interest to the medical and scientific community in therapeutic and other contexts. Although, to date, there has been no direct assessment of the response of human tissue to LDIR when exposure is under normal physiologic conditions of intact three-dimensional architecture, vasculature, and cell-cell contacts (between epithelial cells and between epithelial and stromal cells). Experimental Design: In this article, we present the first data on the response of human tissue exposed in vivo to LDIR with precisely controlled and calibrated doses. We evaluated transcriptomic responses to a single exposure of LDIR in the normal skin of men undergoing therapeutic radiation for prostate cancer (research protocol, Health Insurance Portability and Accountability Act^compliant, Institutional Review Board^approved). Using newly developed biostatistical tools that account for individual splice variants and the expected variability of temporal response between humans even when the outcome is measured at a single time, we show a dose-response pattern in gene expression in a number of pathways and gene groups that are biologically plausible responses to LDIR. Results: Examining genes and pathways identified as radiation-responsive in cell culture models, we found seven gene groups and five pathways that were altered in men in this experiment.These included the Akt/phosphoinositide-3-kinase pathway, the growth factor pathway, the stress/ apoptosis pathway, and the pathway initiated by transforming growth factor-h signaling, whereas gene groups with altered expression included the keratins, the zinc finger proteins and signaling molecules in the mitogen-activated protein kinase gene group.We show that there is considerable individual variability in radiation response that makes the detection of effects difficult, but still feasible when analyzed according to gene group and pathway. Conclusions: These results show for the first time that low doses of radiation have an identifiable biosignature in human tissue, irradiated in vivo with normal intact three-dimensional architecture, vascular supply, and innervation. The genes and pathways show that the tissue (a) does detect the injury, (b) initiates a stress/inflammatory response, (c) undergoes DNA remodeling, as suggested by the significant increase in zinc finger protein gene expression, and (d) initiates a ''pro-survival'' response. The ability to detect a distinct radiation response pattern following LDIR exposure has important implications for risk assessment in both therapeutic and national defense contexts.Although there is increasing concern and interest on the effects of low-dose low -linear energy transfer ionizing radiation (LDIR) in humans, particularly with respect to secondary radiation carcinogenesis following therapeutic radiation, there exists no direct evidence that do...
Purpose-The in vivo effects of low-dose low linear energy transfer ionizing radiation on healthy human skin are largely unknown. Using a patient-based tissue acquisition protocol, we have performed a series of genomic analyses on the temporal dynamics over a 24-hour period to determine the radiation response after a single exposure of 10 cGy.Methods and Materials-RNA from each patient tissue sample was hybridized to an Affymetrix Human Genome U133 Plus 2.0 array. Data analysis was performed on selected gene groups and pathways.Results-Nineteen gene groups and seven gene pathways that had been shown to be radiation responsive were analyzed. Of these, nine gene groups showed significant transient transcriptional changes in the human tissue samples, which returned to baseline by 24 hours postexposure.Conclusions-Low doses of ionizing radiation on full-thickness human skin produce a definable temporal response out to 24 hours postexposure. Genes involved in DNA and tissue remodeling, cell cycle transition, and inflammation show statistically significant changes in expression, despite variability between patients. These data serve as a reference for the temporal dynamics of ionizing radiation response following low-dose exposure in healthy full-thickness human skin.
Current translational human studies are moving in the direction of concurrent genomic and proteomic analysis using small clinical samples. Skin tissue, although easily accessible, is difficult to process owing to its natural resistance to mechanical shearing and high levels of RNases and proteases. Currently, these complications result in degraded RNA samples with variable yield. We have developed a method of sequential extraction of high quality RNA and protein from a single 3 mm full thickness skin punch biopsy. This method yields 1-2 microg of RNA and 150 microg of protein, which is usable in many sensitive downstream applications including microarray, quantitative real-time PCR, two-dimensional gel electrophoresis and Western blot analysis.
Human exposure to arsenic and ionizing radiation occur environmentally at low levels. While the human health effects of arsenic and ionizing radiation have been examined separately, there is little information regarding their combined effects at doses approaching environmental levels. Arsenic toxicity may be affected by concurrent ionizing radiation especially given their known individual carcinogenic actions at higher doses. We found that keratinocytes responded to either low dose arsenic and/or low dose ionizing radiation exposure, resulting in differential proteomic expression based on 2DGE, immunoblotting and statistical analysis. Seven proteins were identified that passed a rigorous statistical screen for differential expression in 2DGE and also passed a strict statistical screen for follow-up immunoblotting. These included: α-enolase, epidermal-fatty acid binding protein, heat shock protein 27, histidine triad nucleotide-binding protein 1, lactate dehydrogenase A, protein disulfide isomerase precursor and S100A9. Four proteins had combined effects that were different than would be expected based on the response to either individual toxicant. These data demonstrate a possible reaction to the combined insult that is substantially different from that of either separate treatment. Several proteins had different responses than what has been seen from high dose exposures, adding to the growing literature suggesting that the cellular responses to low dose exposures are distinct.
20018 Background: As intensity modulated radiation therapy techniques are increasingly utilized to treat cancer, the area of normal tissue exposed to ionizing radiation is increasing. The biologic risks associated with this normal tissue low dose exposure (LDIR) are fundamentally unknown and of concern to cancer survivors following therapy. Current modeling for health regulations presupposes a linear, no-threshold model of radiation effects, which estimates the effect and risk at low dose by extrapolation from measured effects at high doses. Cell culture models of ionizing radiation (RT) exposure show variable effects, not consistent with a linear dose-response relationship. We therefore undertook the first study to our knowledge of transcriptional effects of LDIR over time in vivo in solid tissue in humans. Methods: Tissue was collected at pre-RT, 3, 8, and 24 hours post-IR at sites receiving 10cGy. Transcriptional response at 3 and 8 hours were compared to the 0 and 24 hour time points. If transcripts are up regulated or down regulated at 3 and 8 hours compared with 0 and 24 hours, we have detected a transient response. The method of Rocke (2005), which was designed to detect differentially expressed gene groups using the responses of multiple probe sets corresponding to gene groups, was used to allow us to test whether there is differential expression for each patient separately, as well as for all the patients together. Results: Significant (p < 0.05) transient up regulation was shown in zinc finger proteins, keratins, BMP receptors, BAG, cyclins and BCL 6. Down regulation was detected in TNF, protein disulfide isomerase, interleukins, heat shock proteins, and S100. Nine gene groups did not show significant change; however, the number of significant gene groups (11) far exceeds the number expected by chance (2). In most cases in which a gene group was shown to be transiently altered, the tests of individual patients showed that most or all of the individuals also had differential expression of the same type. Conclusions: We have shown that it is possible to detect transient responses to LDIR in vivo in humans, and have identified eleven gene groups that demonstrate transient changes, as measured by a statistically principled analysis method. No significant financial relationships to disclose.
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