Transcriptional response of kidney tissue after 177Lu-octreotate administration in mice

AbstractIntroductionThe kidneys are one of the main dose limiting organs in 177Lu-octreotate therapy of neuroendocrine tumors. Therefore, biomarkers for radiation damage would be of great importance in this type of therapy. The purpose of this study was to investigate the absorbed dose dependency on early transcriptional changes in the kidneys from 177Lu-octreotate exposure.MethodsFemale Balb/c nude mice were i.v. injected with 1.3, 3.6, 14, 45 or 140MBq 177Lu-octreotate. The animals were killed 24h after injection followed by excision of the kidneys. The absorbed dose to the kidneys ranged between 0.13 and 13Gy. Total RNA was extracted from separated renal tissue samples, and applied to Illumina MouseRef-8 Whole-Genome Expression Beadchips to identify regulated transcripts after irradiation. Nexus Expression 2.0 and Gene Ontology terms were used for data processing and to determine affected biological processes.ResultsDistinct transcriptional responses were observed following 177Lu-octreotate administration. A higher number of differentially expressed transcripts were observed in the kidney medulla (480) compared to cortex (281). In addition, 39 transcripts were regulated at all absorbed dose levels in the medulla, compared to 32 in the cortex. Three biological processes in the cortex and five in the medulla were also shared by all absorbed dose levels. Strong association to metabolism was found among the affected processes in both tissues. Furthermore, an association with cellular and developmental processes was prominent in kidney medulla, while transport and immune response were prominent in kidney cortex.ConclusionSpecific biological and dose-dependent responses were observed in both tissues. The number of affected transcripts and biological processes revealed distinct response differences between the absorbed doses delivered to the tissues

Signaltransduktion und Genexpression in humanen Zellen nach Exposition mit ionisierender Strahlung

Auf Weltraummissionen sind Astronauten einem erhöhten Strahlenrisiko ausgesetzt, das in Dosis und Strahlenspektrum unterschiedlich zur terrestrischen Strahlenexposition ist. Um für Langzeitmissionen das effektive Gesundheitsrisiko abschätzen und medizinische Gegenmaßnahmen entwickeln zu können, sind umfassende Kenntnisse über die zelluläre Strahlenantwort unerlässlich. Im Rahmen dieser Arbeit wurden zentrale Aspekte der NF-κBSignaltransduktion und Genexpression nach Exposition mit dicht und dünn ionisierender Strahlung untersucht. Ein Schadensprofil von DNADoppelstrangbrüchen in HEK-pNF-κB-d2EGFP/Neo L2 nach Strahlenexposition konnte mittels γH2A.X-Immunfluoreszenzfärbung erhoben werden. Die DNADoppelstrangbruch- Reparatur konnte auch mittels Pulsfeld-Gelelektrophorese nachgewiesen und durch Wortmannin inhibiert werden. In MEF-NEMO-/- konnte ein verlängerter G2-Arrest aufgrund des inhibierten kanonischen NF-κBSignalweges nachgewiesen werden. Bei p53-Inhibition durch Pifithrin-α in A549 konnte ein Hinweis auf einen zeitverzögerten, p53-unabhängigen Signalweg zur Induktion des G2-Arrests nach Strahlenexposition erbracht werden. Die Relevanz des jeweiligen Signalwegtyps in der NF-κB-abhängigen Genaktivierung wurde in HEK-pNF-κB-d2EGFP/Neo L2 mittels d2EGFP-Reporter und chemischer Inhibition der Schlüsselkomponenten ATM (KU-55933) und Proteasom (MG-132) untersucht: Die Aktivierung des atypischen NF-κB-Signalweges in Abhängigkeit von der relativen biologischen Wirksamkeit der Strahlung konnte hierbei gezeigt werden. Die nukleäre Translokation von p65/RelA konnte sowohl nach dicht als auch nach dünn ionisierender Bestrahlung über Immunfluoreszenzfärbung visualisiert werden. Eine Genexpressionsanalyse mittels qRT-PCR wurde durchgeführt, wobei eine Hochregulation von nfkbia etwa 2 h nach Exposition mit dicht ionisierender Strahlung festgestellt werden konnte

Systemic effects after ionizing radiation exposure: Genome-wide transcriptional analysis of mouse normal tissues exposed to (211)At, (131)I, or 4 MV photon beam

The radionuclides ¹³¹I and ²¹¹At are used or proposed for a variety of cancer treatments. Both radiohalogens exhibit considerable uptake in the thyroid gland and–to a lower degree–in various other tissues if they are unbound or released from a cancer-targeting agent. The resulting differential exposure throughout the body introduces two paradigms when studying effects in non-thyroid tissues in vivo: 1) low-dose effects of the organ, and 2) systemic effects due to a dominantly exposed regulatory organ. Normal tissue response is an important parameter in risk assessment in order to give the highest possible absorbed dose to malignant tumors while minimizing detrimental side effects in healthy tissue. The aim of this work was to increase fundamental knowledge of normal tissue responses to differential ionizing radiation exposure in vivo and to evaluate key findings regarding circadian rhythm and data convolution. Female BALB/c nude mice were used as a model system and the kidney cortex, kidney medulla, liver, lungs, spleen, and thyroid were studied using RNA microarray technology. Genome-wide transcriptional regulation and basically all analytical endpoints studied were tissue-specific. In various tissues, the <i>Angptl4</i>, <i>Per1</i> and <i>Per2</i>, and <i>Tsc22d3</i> genes may be potential biomarkers for ²¹¹At exposure (Papers I, II). In the thyroid, the <i>Klk1</i> gene-family may serve as biomarker candidates for ¹³¹I exposure (Paper V). Similarity in the extent of regulation irrespective of absorbed dose level generated a hypothesis on thyroid-dependent systemic effects in non-thyroid tissues, which was supported by gene signature and pathway analysis (Papers I, III). Results from partial body irradiation with 4 MV photon beams confirmed the hypothesis (Paper IV). Circadian rhythm affected the extent and quality of regulation in a tissue-specific manner, but key findings showed certain robustness to diurnal variation (Paper V). Deconvolution of microarray data increased detection rate of significantly differentially expressed transcripts in thyroid data, but also confirmed key results derived from convoluted data (Paper VI). In conclusion, low-dose exposure, systemic effects, and circadian rhythm have a pronounced impact on normal tissue response in vivo and should be considered for more accurate risk assessment in radionuclide therapy

Microarray studies on 211At administration in BALB/C nude mice indicate systemic effects on transcriptional regulation in nonthyroid tissues

Targeted a-therapy is a promising treatment option for various types of malignant tumors. Radiolabeled cancer-seeking agents, however, undergo degradation, resulting in a certain percentage of free radionuclide in the body. The radiohalogen 211At accumulates in various tissues, with specifically high uptake in the thyroid. When normal thyroid function is disturbed because of ionizing radiation (IR) exposure, deleterious effects can occur in tissues that depend on thyroid hormone (TH) regulation for normal physiologic function. However, knowledge of systemic effects is still rudimentary. We previously reported similarities in transcriptomic regulation between the thyroid and other tissues despite large differences in absorbed dose from 211At. Here, we present supportive evidence on systemic effects after 211At administration. Methods: Expression microarray data from the kidney cortex and medulla, liver, lungs, and spleen were used from previous studies in which mice were intravenously injected with 0.064-42 kBq of 211At and killed after 24 h or injected with 1.7 kBq of 211At and killed after 1, 6, or 168 h. Controls were mock-treated and killed after 24 h. Literature-based gene signatures were used to evaluate the relative impact from IR- or TH-induced regulation. Thyroid- and TH-associated upstream regulators as well as thyroid-related diseases and functions were generated using functional analysis software. Results: Responses in IR- or TH-associated gene signatures were tissuespecific and varied over time, and the relative impact of each gene signature differed between the investigated tissues. The liver showed a clear dominance of TH-responding genes. In the kidney cortex, kidney medulla, and lungs, the TH-associated signature was detected to at least an extent similar to the IR-associated signature. The spleen was the single tissue showing regulation of only IR-associated signature genes. Various thyroid-associated diseases and functions were inferred from the data: L-triiodothyronine, TH, TH receptor, and triiodothyronine (reverse) were inferred as upstream regulators with differences in incidence and strength of regulation depending on tissue type. Conclusion: These findings indicate that transcriptional regulation in various nonthyroid tissues was-in part-induced by thyroid (hormone)-dependent signaling. Consideration of the systemic context between tissues could contribute to normal tissue risk assessment and planning of remedial measures

Circadian rhythm influences genome-wide transcriptional responses to I-131 in a tissue-specific manner in mice

Background: Circadian variation of gene expression is often neglected when ionizing radiation-induced effects are studied, whether in animal models or in cell culture. This study characterized diurnal variation of genome-wide transcriptional regulation and responses of potential biomarkers and signature genes in normal mouse tissues at 24 h after i.v. administration of I-131. Methods: Female BALB/c nude mice were i.v. injected with 90 kBq I-131 at 9: 00 a.m., 12: 00 p.m., or 3: 00 p.m. and killed after 24 h (n = 4/group). Paired control groups were mock-treated (n = 3-4/group). The kidneys, liver, lungs, spleen, and thyroid were excised, snap-frozen, and stored at -80 degrees C until extraction of total RNA. RNA microarray technology was used for genome-wide expression analysis. Enriched biological processes were categorized after cellular function. Signature genes for ionizing radiation and thyroid hormone-induced responses were taken from the literature. Absorbed dose was estimated using the Medical Internal Radiation Dose (MIRD) formalism. Results: The thyroid received an absorbed dose of 5.9 Gy and non-thyroid tissues received 0.75-2.2 mGy over 24 h. A distinct peak in the total number of significantly regulated transcripts was observed at 9: 00 a. m. in the thyroid, but 3 h later in the kidney cortex, kidney medulla, and liver. Transcriptional regulation in the lungs and spleen was marginal. Associated cellular functions generally varied in quality and response strength between morning, noon, and afternoon. In the thyroid, 25 genes were significantly regulated at all investigated times of day, and 24 thereof showed a distinct pattern of pronounced down-regulation at 9: 00 a. m. and comparatively weak up-regulation at later times. Eleven of these genes belonged to the species-specific kallikrein subfamily Klk1b. Responses in signature genes for thyroid hormone-induced responses were more frequent than for ionizing radiation, and trends persisted irrespective of time of day. Conclusion: Diurnal variation of genome-wide transcriptional responses to 90 kBq I-131 was demonstrated for the thyroid, kidney cortex and medulla, and liver, whereas variation was only marginal in the lungs and spleen. Overall, significant detection of potential biomarkers and signature genes was validated at each time of day, although direction of regulation and fold-change differed between morning, noon, and afternoon. These findings suggest that circadian rhythm should be considered in radiation research and that biological and analytical endpoints should be validated for circadian robustness

Non-targeted transcriptomic effects upon thyroid irradiation: similarity between in-field and out-of-field responses varies with tissue type

Non-targeted effects can induce responses in tissues that have not been exposed to ionizing radiation.Despite their relevance for risk assessment, few studies have investigated these effects in vivo. Inparticular, these effects have not been studied in context with thyroid exposure, which can occur e.g.during irradiation of head and neck tumors. To determine the similarity between in-field and out-offieldresponses in normal tissue, we used a partial body irradiation setup with female mice where thethyroid region, the thorax and abdomen, or all three regions were irradiated. After 24h, transcriptionalregulation in the kidney cortex, kidney medulla, liver, lungs, spleen, and thyroid was analyzed usingmicroarray technology. Thyroid irradiation resulted in transcriptional regulation in the kidney medullaand liver that resembled regulation upon direct exposure of these tissues regarding both strength ofresponse and associated biological function. The kidney cortex showed fewer similarities between thesetups, while the lungs and spleen showed little similarity between in-field and out-of-field responses.Interestingly, effects were generally not found to be additive. Future studies are needed to identifythe molecular mechanisms that mediate these systemic effects, so that they may be used as targets tominimize detrimental side effects in radiotherapy

Comparative Analysis of Transcriptional Gene Regulation Indicates Similar Physiologic Response in Mouse Tissues at Low Absorbed Doses from Intravenously Administered At-211

(211)At is a promising therapeutic radionuclide because of the nearly optimal biological effectiveness of emitted α-particles. Unbound (211)At accumulates in the thyroid gland and in other vital normal tissues. However, few studies have been performed that assess the (211)At-induced normal-tissue damage in vivo. Knowledge about the extent and quality of resulting responses in various organs offers a new venue for reducing risks and side effects and increasing the overall well-being of the patient during and after therapy. METHODS: Female BALB/c nude mice were injected intravenously with 0.064-42 kBq of (211)At or mock-treated, and the kidneys, liver, lungs, and spleen were excised 24 h after injection. A transcriptional gene expression analysis was performed in triplicate using RNA microarray technology. Biological processes associated with regulated transcripts were grouped into 8 main categories with 31 subcategories according to gene ontology terms for comparison of regulatory profiles. RESULTS: A substantial decrease in the total number of regulated transcripts was observed between 0.64 and 1.8 kBq of (211)At for all investigated tissues. Few genes were differentially regulated in each tissue at all absorbed doses. In all tissues, most of these genes showed a nonmonotonous dependence on absorbed dose. However, the direction of regulation generally remained uniform for a given gene. Few known radiation-associated genes were regulated on the transcriptional level, and their expression profile generally appeared to be dose-independent and tissue-specific. The regulatory profiles of categorized biological processes were tissue-specific and reflected the shift in regulatory intensity between 0.64 and 1.8 kBq of (211)At. The profiles revealed strongly regulated and nonregulated subcategories. CONCLUSION: The strong regulatory change observed between 0.64 and 1.8 kBq is hypothesized to result not only from low-dose effects in each tissue but also from physiologic responses to ionizing radiation-induced damage to, for example, the (211)At-accumulating thyroid gland. The presented results demonstrate the complexity of responses to radionuclides in vivo and highlight the need for further research to also consider physiology in ionizing radiation-induced responses