Abstract
The aim of this study was to examine the induction of oxidative stress and apoptosis-associated gene expression profiles in retina after proton irradiation exposure at 0.5 to 4 Gy. Materials and methods: One eye of each Sprague-Dawley rat (6 per group) was irradiated with a conformal proton beam to total doses of 0, 0.5, 1 and 4 Gy. Retinal tissues were isolated for characterization of gene expression profiles 6 hours after proton radiation. Results: For oxidative stress, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly up-regulated (Fmo2, Gpx2, Noxa1 and Sod3) compared to controls. Several important genes involved in the initiation or activation of apoptotic signaling pathways were significantly up-regulated following irradiation (Fas, Faslg, Trp63 and Trp73). TUNEL assay and caspase-3 immunocytochemical analysis revealed increased apoptotic immunoreactivity following irradiation. Conclusion: The data revealed that exposure to proton radiation induced oxidative stress-associated apoptosis. In response to ionizing radiation, the expression of genes involved in pathways mediating apoptosis may be differentially regulated in different dose regimens.
The eye is a unique organ because it is relatively unprotected and is constantly exposed to radiation, atmospheric oxygen, environmental chemicals, and physical abrasion. Concern exists, therefore, about possible radiation damage from occupational exposure and medical procedures.
Radiation-induced oxidative stress occurs via reactive oxygen species (ROS), produced as a consequence of irradiation of biological water (1). Oxidative stress-induced ocular tissue damage resulting from ROS has been associated with a variety of pathological conditions (2-4). Previous studies have shown that oxidative stress is involved in the pathogenesis of radiation-induced retinal damage (5). In many cases, cell death induced by ionizing radiation has been identified as occurring via the process of apoptosis (6, 7). Photoreceptor death by apoptosis is central in the pathology of most forms of retinal degeneration (8). Photoreceptor loss causes irreversible blindness in many retinal diseases (9). Radiation-induced photoreceptor loss was also evident in our previous study (10). However, as increased ROS and reduced antioxidant activity are linked with retinal changes, the underlying mechanisms invoked by retinal cells to regulate radiation-induced oxidative stress are poorly understood; To date, few reports are available on proton radiation-induced changes in gene expression relating to oxidative stress and apoptosis in retinal cells. Thus, the details of cellular mechanisms of radiation-induced retinal cell apoptosis remain unclear. Characterization of radiation-induced marker genes for oxidative stress and apoptosis may provide insight into pathways governing the outcome of differential doses of radiation on the retina.
The purpose of the present study was to investigate the effect of proton irradiation on apoptotic and oxidative stress-associated gene expression profiles in a rat retinal model.
Materials and Methods
Animals and experimental design. Male Sprague-Dawley rats (n=24, 6 per group), each weighing approximately 250 g, were purchased locally (Charles River Breeding Lab, Hollister, CA, USA) and maintained, three per cage, under constant ambient temperature of 18.3°C with a 12-hour day/night cycle. Commercial pellet chow and water were available ad libitum. The animals were observed post-irradiation for signs of radiation-related toxicity. Euthanasia was performed 6 hours after irradiation for analyses. The Loma Linda University Animal Care Facility is fully accredited by the American Association for Accreditation of Laboratory Animal Care and the protocol was approved by the Institutional Animal Care and Use Committee.
Proton radiation. Animals were lightly anesthetized with isoflurane (Halocarbon Laboratories, NJ, USA; 4% induction, 1.5% maintenance). One eye of each rat in the appropriate groups was irradiated with a modulated and shaped 100-MeV conformal proton beam using a previously reported technique (11). Rats were positioned in the path and field localization was achieved using a 0.8 cm diameter light field projected to the (iso) center. The left eye of each rat received a single dose of either 0 (control), 0.5, 1, or 4 Gy, delivered at an average dose rate of 7 Gy/min. A polycarbonate range shifter reduced the range of protons to 0.9 cm in tissue using a stepped propeller. The beam yielded a uniform dose distribution across the irradiated eye at the 90% isodose line.
Eye and retina collection. At euthanasia, three retinas of left eyes from each group were isolated for RNA microarray analysis. The retinal tissue from each animal was placed individually in sterile cryovials, snap frozen in liquid nitrogen and kept at −80°C prior to use. Three left eyes from each subset of group were used for immunohistochemistry. Isolated eyes were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight, rinsed with PBS, infiltrated overnight with 30% sucrose in PBS at 4oC, and then embedded in Optimal Cutting Temperature (OCT) (Sakura, Torrance, CA) and frozen at −80°C.
RNA purification microarrays. RNA extraction and polymerase chain reaction (PCR) array services were performed at SABiosciences service core (Frederick, MD, USA). RNA was extracted from each frozen retina by first disrupting tissues using a ceramic bead-based tissue disruption method on a FastPrep FP120, Bio101 homogenizer (Savant Instruments, Inc., Nassau-Suffolk, NY, USA), followed by phenol/chloroform extraction combined with isopropanol precipitation.
For PCR array service, RNA was quantitated with NanoDrop spectrophotometer, and RNA quality analyzed using Agilent Bioanalyzer with RNA Nano Chip 6000 (Agilent Technologies, Santa Ana, CA, USA). Reverse transcription was performed with SABiosciences' RT2 First Strand Kit (C-03) which contained an effective genomic DNA elimination step and a built-in external RNA control. RNA input was 1000 ng per RT reaction. cDNA template was then run on appropriate PCR arrays in an ABI 7900HT sequence detection system. PCR reactions were performed to evaluate expression of 84 genes using RT2 Prolifer™ PCR array PARN-12 (Rat Apoptosis SuperArray) and PARN-065 (Rat Oxidative Stress SuperArray). Data analysis was performed using SABiosciences' PCR array data analysis software. Relative changes in gene expression were calculated using the comparative threshold cycle (Delta Delta Ct, DDCt) method. This method first subtracts the Ct (threshold cycle number) of the gene-average Ct of the 5 housekeeping genes on the array (Rplp1, Hprt, Prl13a Ldha and Actb) to normalize for the amount of RNA per sample. Finally the DDCt was calculated as the difference between the normalized average Ct of the irradiated group and the normalized average Ct of the non-irradiated control group. This DDCt value was raised to the power of 2 to calculate the degree of change. The genes of interest included those involved in oxidative stress and apoptosis. Three rat retinas per group were used for this analysis.
Photoreceptor cell apoptosis detection by TUNEL assay and activated caspase-3 immunohistochemical labeling. In the present study, apoptosis was evaluated 6 hours post irradiation. Sections of 5 μm bisecting the optic disc superiorly to inferiorly were cut on a microtome (Leica Inc., Deerfield, IL, USA). Paraffin sections were deparaffinized in Histo-clear, then permeabilized in 0.3% TritonX-100. Retinal tissues were subjected to terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) using DeadEnd™ Fluorometric TUNEL system kit (Promega, Corp., Madison, WI, USA). As an additional way to confirm the apoptotic process in the retina, sections were immunocytochemically labeled with Anti-ACTIVE® Caspase-3 polyclonal antibody (Promega) specific for activated caspase-3. Sections were then incubated with anti-active caspase-3 primary antibody at room temperature for 2 hours followed by a donkey anti-rabbit IgG fluorescent-conjugated secondary antibody (Invitrogen Corp. Carlsbad, CA, USA) for 2 hours at room temperature and counterstained with propidium iodide (PI). Five random fields on one tissue section of the retina (horizontal, central sections, passing through the optic nerve head) were examined at 300 μm nasally and temporally of the optic nerve using an Olympus IX81 microscope (Olympus America Inc., Center Valley, PA, USA). TUNEL-positive cells were identified by green fluorescence; the nuclei of photoreceptor were counterstained with PI (red). For each field, the numbers of TUNEL-positive cells and total nuclei in the outer nuclear (ONL) and inner nuclear layer (INL) were counted. Percentages of apoptotic-to-total nuclei were averaged across three sections per animal (separated by 50 μm) to provide a single value for the entire retina.
To obtain the resulting activated caspase-3 immunoreactivity, fluorescence intensity was measured on 5 randomly selected fields on each section and calculated using Image J 1.41 software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/Java). Once the green channel was separated from the red channel in an image, fluorescence intensities from the areas of interest were measured using the integral/density feature in the Image J program and data were extracted and averaged within the group.
Statistical analyses. Results were subjected to statistical analyses including one-way analysis of variance (ANOVA) and Tukey's HSD (honestly significant difference) test using SigmaStat™ version 3.5 software (SPSS Inc., Chicago, IL, USA). A p-value of <0.05 was selected to indicate significant differences among groups.
Results
Gene expression. The genes that were statistically significantly (p<0.05) altered between irradiated and control groups by >1.5-fold are presented in a tabular format: Table I lists genes of the oxidative stress-related pathway. Table II contains the list of apoptosis-associated genes. For the 84 oxidative stress genes that were evaluated, significant dose-dependent changes in irradiated retina were noted when compared with the 0 Gy-treated group (p<0.05): 5 genes were up-regulated and 2 were down-regulated at 0.5 Gy; 8 were up-regulated and none were down-regulated at 1 Gy; 3 were up-regulated and 1 was down-regulated at 4 Gy. Several genes playing a central role in regulating ROS production under conditions of oxidative stress were up-regulated. These include: flavin-containing monooxygenase 2 (Fmo2), glutathione peroxidase 2 (Gpx2), NADPH oxidase activator 1 (Noxa1), and superoxide dismutase 3 (Sod3).
For the apoptotic gene profile based on the 84 evaluated genes, the data revealed that 7 genes were up-regulated and 1 was down-regulated at 0.5 Gy; at 1 Gy, 10 were up-regulated and 2 were down-regulated; whereas, 5 were up-regulated and none were down-regulated at 4 Gy (p<0.05 vs.0 Gy; Table II). Several important genes involved in the initiation or activation of apoptotic signaling pathways were significantly up-regulated. They included: TNF receptor superfamily, member 6 (Fas), Fas ligand (Faslg), transformation related protein 63 (Trp63), and Trp73. Genes participating in the caspase cascade were also up-regulated: apoptotic peptidase activating factor 1 (Apaf), Bcl2-associated X protein (Bax), caspases 3, 8 and 14 (Casp3, Casp8 and Casp14).
Apoptosis assessment following radiation. To investigate whether radiation induced retinal cell loss via apoptosis, a TUNEL assay and caspases-3 immunohistochemical labeling of retinal sections were performed. At 6 hours' post irradiation, greater numbers of apoptotic cells were observed in the INL and ONL after 1 Gy (Figure 1c) and 4 Gy (Figure 1d) exposure compared to controls (Figure 1a) or retinas that received 0.5 Gy (Figure 1b). Only sparse TUNEL-positive cells were found in the photoreceptor layer of eyes that received 0.5 Gy. 35.5±7.2% and 21.8± 4.3% of the cells that received 4 Gy and 1 Gy were labeled TUNEL positively in ONL and INL compared to age-matched controls (p<0.05) respectively. Our assessment (Figure 2) also revealed that the immunoreactivity for activated caspase-3 after 1 and 4 Gy of proton irradiation exposure at 6 hours were significantly higher than that of controls and the 0.5 Gy irradiated group (p<0.01).
Discussion
Ionizing radiation induces the production of ROS, which play an important causative role in apoptotic-mediated cell death. Many studies have documented oxidative stress associated with radiation-induced apoptosis (12, 13). Our aim was to characterize proton radiation-induced modulation of gene expression in rat retina. This study demonstrated that radiation significantly induced changes in oxidative stress and apoptosis-associated gene expression in the rat retina at doses as low as 0.5 Gy. There was a significant increase in apoptotic cell death measurable at 1 Gy. Our data are in agreement with other studies of the acute effects of ionizing radiation on the rat retina (14, 15).
The induction of oxygen radical formation is a major mechanism by which radiation induces DNA damage (16). Free radicals can also initiate a variety of cellular signal transduction pathways that lead to damage beyond the repair capabilities of the cell (17). In this study, many genes in the oxidase or peroxidase families were up-regulated following radiation; these included Fmo2, Duox1, Mpo, Prdx4, and Tpo. Interestingly, a recent study showed thyroid peroxidase-induced Bcl-xL gene expression, an anti-apoptotic member of the Bcl-2 family (18). The data suggest possible associations between oxidative stress and apoptosis. Txnrd1, the gene for thioredoxin reductase was also up-regulated at 1 Gy. This is an important selenoprotein that aids in the maintenance of cellular redox balance and regulates several redox-dependent processes in the apoptosis pathway, cell proliferation and differentiation (19). Our data indicated that Sod3 (superoxide dismutase 3, extracellular) was significantly up-regulated following irradiation at 1 Gy. Superoxide dismutases are a ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Sod3 has been shown to play an important role in scavenging ROS. A previous study reported that increased levels of SOD3 protein prevented the degradation of NO by oxygen radicals (20).
The presence of oxidative stress in the irradiated retina was evident at a dose as low as 0.5 Gy. We saw the most significant regulation of genes in retinal samples that received 1 Gy of proton radiation compared to samples that received lower (0.5 Gy) or higher doses (4 Gy). The considerable reduction of gene expression following the 4 Gy of proton radiation was probably due to the increased cell death at this high dose, or alternatively the inability of lethally irradiated cells to regulate their response to injury. Overall, the gene expression patterns in both arrays showed a striking dependence on the total dose that was delivered to the retinal tissue.
The up-regulation of oxidative stress genes is an early marker of retinal degeneration. The presence of markers for apoptosis in this study supports the hypothesis that apoptotic mechanisms are involved in retinal cell death. Radiation-induced apoptosis can take place through several routes. There are two major mechanisms for apoptosis in the retina. They involve the Fas/Fas-L interaction system and the p53 cascade (21-23). In our study, the genes encoding Fas and Fas ligand proteins were significantly up-regulated by 0.5 and 1 Gy of proton irradiation compared to control samples. The data support the possible involvement of Fas in this form of apoptosis occurring in the retina. Thus, Fas/FasL interactions may play an important role in identifying and/or eliminating damaged cells after low-dose irradiation and possibly other forms of injury. In contrast, at 4 Gy, p53 family members Trp63 and Trp73 genes were significantly up-regulated, suggesting that p53, not Fas/Fas-L, plays a major role at relative high doses of radiation-induced retinal cell apoptosis. P53 is activated and up-regulated following a range of insults including DNA damage, hypoxia, oxidative stress, and viral infections (24). The p53 gene product acts as a transcription factor, inducing apoptosis by modulating specific downstream target genes (25). Surprisingly, in our present study, although p53 family members Trp63 and Trp73 were up-regulated, Trp53 was not significantly up-regulated. The failure of detection of this gene at 6 hours post irradiation could point to its early involvement in apoptosis following irradiation. Previous study demonstrated that expression of p53 mRNA in RPE cells increased significantly within 2 hours after exposure to ionizing radiation (25). Recent studies reported that over expression of Trp73 can activate the transcription of p53-responsive genes and can induce apoptosis (27). At 0.5 Gy, baculoviral inhibitor of apoptosis repeat-containing 6 (Birc 6) gene was significantly down-regulated. The Birc6 gene encodes an inhibitor of apoptosis; this gene has been shown to regulate mitochondrial pathway of apoptosis (28). Down-regulation of Birc6 gene further demonstrated that radiation can induce retinal apoptosis at a dose as low as 0.5Gy. These data indicate that in response to physiological stress induced by ionizing radiation, the expression of genes involved in pathways regulating apoptosis may be differentially regulated at different doses.
The universal biochemical hallmark of apoptotic death is the activation of caspases, which are cysteine proteases characterized by their ability to cleave specific proteins (29). Previously published findings reported that Fas-mediated apoptosis involves caspase-8 upon activation of downstream caspases (30). The findings of our gene expression data are consistent with this. We found that caspase-8 and other caspase genes were significantly up-regulated following radiation exposure. Among them, we measured caspase-14 as being the most highly up-regulated gene following 1 Gy of radiation (an approximate 13-fold increase). Caspase-14 is expressed in a broad range of epithelial cell types including skin, breast, prostate, and stomach (31). Caspase-14 protein was also present at lower levels in the brain, and testis of humans and mice (32). To our knowledge, our study is the first to suggest the presence of caspase-14 in rat retina. Caspase-14 may be involved in engagement of the death receptor in apoptotic pathways. It functions as a downstream signal transducer of cell death. Caspase-14 is processed and activated by caspase-8 and caspase-9 in mice (33). This is supported by our observations that expression changes were detected in both caspase-14 and caspase-8 in response to exposure to 1 Gy protons. Caspase-3 acts as an effector caspase of apoptosis by cleaving cellular substrates and precipitating apoptotic death. Our immunochemical data further support caspase 3 involvement in apoptosis of the retina measured 6 hours' post proton irradiation.
Radiation-induced ROS-meditation of apoptosis is a major oxidative stress pathway in the retina. Our study provides some insight into the cellular mechanisms critical for responsiveness to acute oxidative injury of the retina and may prompt the development of new strategies against ocular damage caused by oxidative stress associated apoptosis.
Acknowledgements
The Authors thank Steve Rightnar for his support on the radiation physics aspects. The study was supported by the National Aeronautics and Space Administration (grant No. NNX08AP21G), the National Medical Technology Testbed (NMTB), and the LLUMC Department of Radiation Medicine.
- Received March 16, 2010.
- Revision received May 31, 2010.
- Accepted June 4, 2010.
- Copyright © 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved