Transient genome-wide transcriptional response to low-dose ionizing radiation in vivo in humans.

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.

Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies.

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 in vivo dose-response to controlled, low-dose low linear energy transfer ionizing radiation exposure.

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-beta 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.

Dosimetry for quantitative analysis of the effects of low-dose ionizing radiation in radiation therapy patients.

We have developed and validated a practical approach to identifying the location on the skin surface that will receive a prespecified biopsy dose (ranging down to 1 cGy) in support of in vivo biological dosimetry in humans. This represents a significant technical challenge since the sites lie on the patient's surface outside the radiation fields. The PEREGRINE Monte Carlo simulation system was used to model radiation dose delivery, and TLDs were used for validation on phantoms and for confirmation during patient treatment. In the developmental studies, the Monte Carlo simulations consistently underestimated the dose at the biopsy site by approximately 15% (of the local dose) for a realistic treatment configuration, most likely due to lack of detail in the simulation of the linear accelerator outside the main beam line. Using a single, thickness-independent correction factor for the clinical calculations, the average of 36 measurements for the predicted 1-cGy point was 0.985 cGy (standard deviation: 0.110 cGy) despite patient breathing motion and other real-world challenges. Since the 10-cGy point is situated in the region of high-dose gradient at the edge of the field, patient motion had a greater effect, and the six measured points averaged 5.90 cGy (standard deviation: 1.01 cGy), a difference that is equivalent to approximately a 6-mm shift on the patient's surface.

Effects of low-dose ionizing radiation on gene expression in human skin biopsies.

PURPOSE: Several investigations have demonstrated that significant biologic effects can occur in animals, animal cells, immortalized human cell lines, and primary human cells after exposure to doses of ionizing radiation in the low-dose, < or =1-10 cGy region (LDIR). However, little information is available as to how these and other observations pertain to human responses to LDIR, though such knowledge is required for reducing the uncertainty of assessing human risks due to these exposures. We therefore undertook these translational studies to begin the development of a unique data set of human cellular responses to LDIR as measured by gene expression changes when exposure occurs to a normal tissue with its complex cellular mixture and three-dimensional architecture. METHODS AND MATERIALS: Using full-thickness human skin resected during esthetic surgery, we obtained biopsy cores and exposed the tissue to LDIR ex vivo. Gene expression changes in five core regulatory genes were assessed by real-time RT-PCR. RESULTS: Results indicate that skin is a good biologic model for assessing LDIR in humans, though meticulous attention to sample processing is necessary. LDIR does produce changes in gene expression, though time- and dose-response relationships may be complex. CONCLUSION: These proof-of-principle studies have provided a crucial initial step toward validation of LDIR risk assessment models in humans. We have demonstrated the feasibility of this approach and provide initial evidence that ionizing radiation exposures as low as 1 cGy are biologically active in human skin.