Effects of Recombinant α1-Microglobulin on Early Proteomic Response in Risk Organs after Exposure to 177Lu-Octreotate.

Recombinant α1-microglobulin (A1M) is proposed as a protector during 177Lu-octreotate treatment of neuroendocrine tumors, which is currently limited by bone marrow and renal toxicity. Co-administration of 177Lu-octreotate and A1M could result in a more effective treatment by protecting healthy tissue, but the radioprotective action of A1M is not fully understood. The aim of this study was to examine the proteomic response of kidneys and bone marrow early after 177Lu-octreotate and/or A1M administration. Mice were injected with 177Lu-octreotate and/or A1M, while control mice received saline or A1M vehicle solution. Bone marrow, kidney medulla, and kidney cortex were sampled after 24 h or 7 d. The differential protein expression was analyzed with tandem mass spectrometry. The dosimetric estimation was based on 177Lu activity in the kidney. PHLDA3 was the most prominent radiation-responsive protein in kidney tissue. In general, no statistically significant difference in the expression of radiation-related proteins was observed between the irradiated groups. Most canonical pathways were identified in bone marrow from the 177Lu-octreotate+A1M group. Altogether, a tissue-dependent proteomic response followed exposure to 177Lu-octreotate alone or together with A1M. Combining 177Lu-octreotate with A1M did not inhibit the radiation-induced protein expression early after exposure, and late effects should be further studied.

Development of a machine learning framework for radiation biomarker discovery and absorbed dose prediction.

Background: Molecular radiation biomarkers are an emerging tool in radiation research with applications for cancer radiotherapy, radiation risk assessment, and even human space travel. However, biomarker screening in genome-wide expression datasets using conventional tools is time-consuming and underlies analyst (human) bias. Machine Learning (ML) methods can improve the sensitivity and specificity of biomarker identification, increase analytical speed, and avoid multicollinearity and human bias. Aim: To develop a resource-efficient ML framework for radiation biomarker discovery using gene expression data from irradiated normal tissues. Further, to identify biomarker panels predicting radiation dose with tissue specificity. Methods: A strategic search in the Gene Expression Omnibus database identified a transcriptomic dataset (GSE44762) for normal tissues radiation responses (murine kidney cortex and medulla) suited for biomarker discovery using an ML approach. The dataset was pre-processed in R and separated into train and test data subsets. High computational cost of Genetic Algorithm/k-Nearest Neighbor (GA/KNN) mandated optimization and 13 ML models were tested using the caret package in R. Biomarker performance was evaluated and visualized via Principal Component Analysis (PCA) and dose regression. The novelty of ML-identified biomarker panels was evaluated by literature search. Results: Caret-based feature selection and ML methods vastly improved processing time over the GA approach. The KNN method yielded overall best performance values on train and test data and was implemented into the framework. The top-ranking genes were Cdkn1a, Gria3, Mdm2 and Plk2 in cortex, and Brf2, Ccng1, Cdkn1a, Ddit4l, and Gria3 in medulla. These candidates successfully categorized dose groups and tissues in PCA. Regression analysis showed that correlation between predicted and true dose was high with R2 of 0.97 and 0.99 for cortex and medulla, respectively. Conclusion: The caret framework is a powerful tool for radiation biomarker discovery optimizing performance with resource-efficiency for broad implementation in the field. The KNN-based approach identified Brf2, Ddit4l, and Gria3 mRNA as novel candidates that have been uncharacterized as radiation biomarkers to date. The biomarker panel showed good performance in dose and tissue separation and dose regression. Further training with larger cohorts is warranted to improve accuracy, especially for lower doses.

Machine intelligence for radiation science: summary of the Radiation Research Society 67th annual meeting symposium.

The era of high-throughput techniques created big data in the medical field and research disciplines. Machine intelligence (MI) approaches can overcome critical limitations on how those large-scale data sets are processed, analyzed, and interpreted. The 67th Annual Meeting of the Radiation Research Society featured a symposium on MI approaches to highlight recent advancements in the radiation sciences and their clinical applications. This article summarizes three of those presentations regarding recent developments for metadata processing and ontological formalization, data mining for radiation outcomes in pediatric oncology, and imaging in lung cancer.

Age and sex effects across the blood proteome after ionizing radiation exposure can bias biomarker screening and risk assessment.

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 h after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6 N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage and repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.

Age-related long-term response in rat thyroid tissue and plasma after internal low dose exposure to 131I.

131I is used clinically for therapy, and may be released during nuclear accidents. After the Chernobyl accident papillary thyroid carcinoma incidence increased in children, but not adults. The aims of this study were to compare 131I irradiation-dependent differences in RNA and protein expression in the thyroid and plasma of young and adult rats, and identify potential age-dependent biomarkers for 131I exposure. Twelve young (5 weeks) and twelve adult Sprague Dawley rats (17 weeks) were i.v. injected with 50 kBq 131I (absorbed dose to thyroid = 0.1 Gy), and sixteen unexposed age-matched rats were used as controls. The rats were killed 3-9 months after administration. Microarray analysis was performed using RNA from thyroid samples, while LC-MS/MS analysis was performed on proteins extracted from thyroid tissue and plasma. Canonical pathways, biological functions and upstream regulators were analysed for the identified transcripts and proteins. Distinct age-dependent differences in gene and protein expression were observed. Novel biomarkers for thyroid 131I exposure were identified: (PTH), age-dependent dose response (CA1, FTL1, PVALB (youngsters) and HSPB6 (adults)), thyroid function (Vegfb (adults)). Further validation using clinical samples are needed to explore the role of the identified biomarkers.

Acknowledging and overcoming barriers to entry into radiation science for women.

PURPOSE: Radiation science is a unique field that brings together various disciplines to understand nature, develop new technologies, and cure diseases. Our field is a prime example of advancement through a diverse pool of competencies. Similarly, studies show that the power of diversity requires proportionate representation of sex and gender, minorities, or other groups. Nevertheless, women are still underrepresented in the radiation sciences, although disparities and underlying mechanisms were first described decades ago. This review summarizes barriers to entry and retention and suggests strategies for overcoming disparities in our field. We also highlight a concerted effort by young professionals to promote the underrepresented and underserved within the radiation science community. CONCLUSION: The radiation science community should avoid losing diverse perspectives among its ranks due to sex bias or gender disparity among others. Through targeted efforts, we can cultivate change and harness the talent of researchers, practitioners, and other professionals for the benefit of scientific progress, health-care improvement, and societal advancement overall.

Avasopasem manganese synergizes with hypofractionated radiation to ablate tumors through the generation of hydrogen peroxide.

Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine- and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide-dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974).

Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

BACKGROUND: Radioiodide (131I) is commonly used to treat thyroid cancer and hyperthyroidis.131I released during nuclear accidents, have resulted in increased incidence of thyroid cancer in children. Therefore, a better understanding of underlying cellular mechanisms behind 131I exposure is of great clinical and radiation protection interest. The aim of this work was to study the long-term dose-related effects of 131I exposure in thyroid tissue and plasma in young rats and identify potential biomarkers. MATERIALS AND METHODS: Male Sprague Dawley rats (5-week-old) were i.v. injected with 0.5, 5.0, 50 or 500 kBq 131I (Dthyroid ca 1-1000 mGy), and killed after nine months at which time the thyroid and blood samples were collected. Gene expression microarray analysis (thyroid samples) and LC-MS/MS analysis (thyroid and plasma samples) were performed to assess differential gene and protein expression profiles in treated and corresponding untreated control samples. Bioinformatics analyses were performed using the DAVID functional annotation tool and Ingenuity Pathway Analysis (IPA). The gene expression microarray data and LC-MS/MS data were validated using qRT-PCR and ELISA, respectively. RESULTS: Nine 131I exposure-related candidate biomarkers (transcripts: Afp and RT1-Bb, and proteins: ARF3, DLD, IKBKB, NONO, RAB6A, RPN2, and SLC25A5) were identified in thyroid tissue. Two dose-related protein candidate biomarkers were identified in thyroid (APRT and LDHA) and two in plasma (DSG4 and TGM3). Candidate biomarkers for thyroid function included the ACADL and SORBS2 (all activities), TPO and TG proteins (low activities). 131I exposure was shown to have a profound effect on metabolism, immune system, apoptosis and cell death. Furthermore, several signalling pathways essential for normal cellular function (actin cytoskeleton signalling, HGF signalling, NRF2-mediated oxidative stress, integrin signalling, calcium signalling) were also significantly regulated. CONCLUSION: Exposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer.

The IRI-DICE hypothesis: ionizing radiation-induced DSBs may have a functional role for non-deterministic responses at low doses.

Low-dose ionizing radiation (IR) responses remain an unresolved issue in radiation biology and risk assessment. Accurate knowledge of low-dose responses is important for estimation of normal tissue risk in cancer radiotherapy or health risks from occupational or hazard exposure. Cellular responses to low-dose IR appear diverse and stochastic in nature and to date no model has been proposed to explain the underlying mechanisms. Here, we propose a hypothesis on IR-induced double-strand break (DSB)-induced cis effects (IRI-DICE) and introduce DNA sequence functionality as a submicron-scale target site with functional outcome on gene expression: DSB induction in a certain genetic target site such as promotor, regulatory element, or gene core would lead to changes in transcript expression, which may range from suppression to overexpression depending on which functional element was damaged. The DNA damage recognition and repair machinery depicts threshold behavior requiring a certain number of DSBs for induction. Stochastically distributed persistent disruption of gene expression may explain-in part-the diverse nature of low-dose responses until the repair machinery is initiated at increased absorbed dose. Radiation quality and complexity of DSB lesions are also discussed. Currently, there are no technologies available to irradiate specific genetic sites to test the IRI-DICE hypothesis directly. However, supportive evidence may be achieved by developing a computational model that combines radiation transport codes with a genomic DNA model that includes sequence functionality and transcription to simulate expression changes in an irradiated cell population. To the best of our knowledge, IRI-DICE is the first hypothesis that includes sequence functionality of different genetic elements in the radiation response and provides a model for the diversity of radiation responses in the (very) low dose regimen.

Radiation-induced genomic instability in breast carcinomas of the Swedish hemangioma cohort.

Radiation-induced genomic instability (GI) is hypothesized to persist after exposure and ultimately promote carcinogenesis. Based on the absorbed dose to the breast, an increased risk of developing breast cancer was shown in the Swedish hemangioma cohort that was treated with radium-226 for skin hemangioma as infants. Here, we screened 31 primary breast carcinomas for genetic alterations using the OncoScan CNV Plus Assay to assess GI and chromothripsis-like patterns associated with the absorbed dose to the breast. Higher absorbed doses were associated with increased numbers of copy number alterations in the tumor genome and thus a more unstable genome. Hence, the observed dose-dependent GI in the tumor genome is a measurable manifestation of the long-term effects of irradiation. We developed a highly predictive Cox regression model for overall survival based on the interaction between absorbed dose and GI. The Swedish hemangioma cohort is a valuable cohort to investigate the biological relationship between absorbed dose and GI in irradiated humans. This work gives a biological basis for improved risk assessment to minimize carcinogenesis as a secondary disease after radiation therapy.

Transcriptional effects of 177Lu-octreotate therapy using a priming treatment schedule on GOT1 tumor in nude mice.

BACKGROUND: 177Lu-octreotate is used for therapy of somatostatin receptor expressing neuroendocrine tumors with promising results, although complete tumor remission is rarely seen. Previous studies on nude mice bearing the human small intestine neuroendocrine tumor, GOT1, have shown that a priming injection of 177Lu-octreotate 24 h before the main injection of 177Lu-octreotate resulted in higher 177Lu concentration in tumor, resulting in increased absorbed dose, volume reduction, and time to regrowth. To our knowledge, the cellular effects of a priming treatment schedule have not yet been studied. The aim of this study was to identify transcriptional changes contributing to the enhanced therapeutic response of GOT1 tumors in nude mice to 177Lu-octreotate therapy with priming, compared with non-curative monotherapy. RESULTS: RNA microarray analysis was performed on tumor samples from GOT1-bearing BALB/c nude mice treated with a 5 MBq priming injection of 177Lu-octreotate followed by a second injection of 10 MBq of 177Lu-octreotate after 24 h and killed after 1, 3, 7, and 41 days after the last injection. Administered activity amounts were chosen to be non-curative, in order to facilitate the study of tumor regression and regrowth. Differentially regulated transcripts (RNA samples from treated vs. untreated animals) were identified (change ≥ 1.5-fold; adjusted p value < 0.01) using Nexus Expression 3.0. Analysis of the biological effects of transcriptional regulation was performed using the Gene Ontology database and Ingenuity Pathway Analysis. Transcriptional analysis of the tumors revealed two stages of pathway regulation for the priming schedule (up to 1 week and around 1 month) which differed distinctly from cellular responses observed after monotherapy. Induction of cell cycle arrest and apoptotic pathways (intrinsic and extrinsic) was found at early time points after treatment start, while downregulation of pro-proliferative genes were found at a late time point. CONCLUSIONS: The present study indicates increased cellular stress responses in the tumors treated with a priming treatment schedule compared with those seen after conventional 177Lu-octreotate monotherapy, resulting in a more profound initiation of cell cycle arrest followed by apoptosis, as well as effects on PI3K/AKT-signaling and unfolded protein response.

Time-dependent transcriptional response of GOT1 human small intestine neuroendocrine tumor after 177Lu[Lu]-octreotate therapy.

INTRODUCTION: Patients with neuroendocrine tumors expressing somatostatin receptors are often treated with 177Lu[Lu]-octreotate. Despite being highly effective in animal models, 177Lu[Lu]-octreotate-based therapies in the clinical setting can be optimized further. The aims of the study were to identify and elucidate possible optimization venues for 177Lu[Lu]-octreotate tumor therapy by characterizing transcriptional responses in the GOT1 small intestine neuroendocrine tumor model in nude mice. METHODS: GOT1-bearing female BALB/c nude mice were intravenously injected with 15 MBq 177Lu[Lu]-octreotate (non-curative amount) or mock-treated with saline solution. Animals were killed 1, 3, 7 or 41 d after injection. Total RNA was extracted from the tumor samples and profiled using Illumina microarray expression analysis. Differentially expressed genes were identified (treated vs. control) and pathway analysis was performed. RESULTS: Distribution of differentially expressed transcripts indicated a time-dependent treatment response in GOT1 tumors after 177Lu[Lu]-octreotate administration. Regulation of CDKN1A, BCAT1 and PAM at 1 d after injection was compatible with growth arrest as the initial response to treatment. Upregulation of APOE and BAX at 3 d, and ADORA2A, BNIP3, BNIP3L and HSPB1 at 41 d after injection suggests first activation and then inhibition of the intrinsic apoptotic pathway during tumor regression and regrowth, respectively. CONCLUSION: Transcriptional analysis showed radiation-induced apoptosis as an early response after 177Lu[Lu]-octreotate administration, followed by pro-survival transcriptional changes in the tumor during the regrowth phase. Time-dependent changes in cell cycle and apoptosis-related processes suggest different time points after radionuclide therapy when tumor cells may be more susceptible to additional treatment, highlighting the importance of timing when administering multiple therapeutic agents.

Hedgehog inhibitor sonidegib potentiates 177Lu-octreotate therapy of GOT1 human small intestine neuroendocrine tumors in nude mice.

BACKGROUND: 177Lu-octreotate can be used to treat somatostatin receptor expressing neuroendocrine tumors. It is highly effective in animal models, but clinical studies have so far only demonstrated low cure rates. Hedgehog inhibitors have shown therapeutic effect as monotherapy in neuroendocrine tumor model systems and might be one option to enhance the efficacy of 177Lu-octreotate therapy. The aim of this study was to determine the therapeutic effect of combination therapy using 177Lu-octreotate and the Hedgehog signaling pathway inhibitor sonidegib. METHODS: GOT1-bearing BALB/c nude mice were treated with either sonidegib (80 mg/kg twice a week via oral gavage), a single injection of 30 MBq 177Lu-octreotate i.v., or a combination of both. Untreated animals served as controls. Tumor size was measured twice-weekly using calipers. The animals were killed 41 d after injection followed by excision of the tumors. Total RNA was extracted from each tumor sample and then subjected to gene expression analysis. Gene expression patterns were compared with those of untreated controls using Nexus Expression 3.0, IPA and Gene Ontology terms. Western blot was carried out on total protein extracted from the tumor samples to analyze activation-states of the Hh and PI3K/AKT/mTOR pathways. RESULTS: Sonidegib monotherapy resulted in inhibition of tumor growth, while a significant reduction in mean tumor volume was observed after 177Lu-octreotate monotherapy and combination therapy. Time to progression was prolonged in the combination therapy group compared with 177Lu-octreotate monotherapy. Gene expression analysis revealed a more pronounced response following combination therapy compared with both monotherapies, regarding the number of regulated genes and biological processes. Several cancer-related signaling pathways (i.e. Wnt/ß-catenin, PI3K/AKT/mTOR, G-protein coupled receptor, and Notch) were affected by the combination therapy, but not by either monotherapy. Protein expression analysis revealed an activation of the Hh- and PI3K/AKT/mTOR pathways in tumors exposed to 177Lu-octreotate monotherapy and combination therapy. CONCLUSIONS: A comparative analysis of the different treatment groups showed that combination therapy using sonidegib and 177Lu-octreotate could be beneficial to patients with neuroendocrine tumors. Gene expression analysis revealed a functional interaction between sonidegib and 177Lu-octreotate, i.e. several cancer-related signaling pathways were modulated that were not affected by either monotherapy. Protein expression analysis indicated a possible PI3K/AKT/mTOR-dependent activation of the Hh pathway, independent of SMO.

Priming increases the anti-tumor effect and therapeutic window of 177Lu-octreotate in nude mice bearing human small intestine neuroendocrine tumor GOT1.

BACKGROUND: 177Lu-[DOTA0, Tyr3]-octreotate (177Lu-octreotate) is used for treatment of patients with somatostatin receptor (SSTR) expressing neuroendocrine tumors. However, complete tumor remission is rarely seen, and optimization of treatment protocols is needed. In vitro studies have shown that irradiation can up-regulate the expression of SSTR1, 2 and 5, and increase 177Lu-octreotate uptake. The aim of the present study was to examine the anti-tumor effect of a 177Lu-octreotate priming dose followed 24 h later by a second injection of 177Lu-octreotate compared to a single administration of 177Lu-octreotate, performed on the human small intestine neuroendocrine tumor cell line, GOT1, transplanted to nude mice. RESULTS: Priming resulted in a 1.9 times higher mean absorbed dose to the tumor tissue per administered activity, together with a reduced mean absorbed dose for kidneys. Priming gave the best overall anti-tumor effects. Magnetic resonance imaging showed no statistically significant difference in tumor response between treatment with and without priming. Gene expression analysis demonstrated effects on cell cycle regulation. Biological processes associated with apoptotic cell death were highly affected in the biodistribution and dosimetry study, via differential regulation of, e.g., APOE, BAX, CDKN1A, and GADD45A. CONCLUSIONS: Priming had the best overall anti-tumor effects and also resulted in an increased therapeutic window. Results indicate that potential biomarkers for tumor regrowth may be found in the p53 or JNK signaling pathways. Priming administration is an interesting optimization strategy for 177Lu-octreotate therapy of neuroendocrine tumors, and further studies should be performed to determine the mechanisms responsible for the reported effects.

Microarray Studies on 211At Administration in BALB/c Nude Mice Indicate Systemic Effects on Transcriptional Regulation in Nonthyroid Tissues.

Targeted α-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 tissue-specific 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.

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. In particular, 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-of-field responses in normal tissue, we used a partial body irradiation setup with female mice where the thyroid region, the thorax and abdomen, or all three regions were irradiated. After 24 h, transcriptional regulation in the kidney cortex, kidney medulla, liver, lungs, spleen, and thyroid was analyzed using microarray technology. Thyroid irradiation resulted in transcriptional regulation in the kidney medulla and liver that resembled regulation upon direct exposure of these tissues regarding both strength of response and associated biological function. The kidney cortex showed fewer similarities between the setups, 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 identify the molecular mechanisms that mediate these systemic effects, so that they may be used as targets to minimize detrimental side effects in radiotherapy.

Gene expression signature in mouse thyroid tissue after (131)I and (211)At exposure.

BACKGROUND: (131)I and (211)At are used in nuclear medicine and accumulate in the thyroid gland and may impact normal thyroid function. The aim of this study was to determine transcriptional profile variations, assess the impact on cellular activity, and identify genes with biomarker properties in thyroid tissue after (131)I and (211)At administration in mice. METHODS: To further investigate thyroid tissue transcriptional responses to (131)I and (211)At administration, we generated a new transcriptional dataset that includes re-evaluated raw intensity values from our previous (131)I and (211)At studies. Differential transcriptional profiles were identified by comparing treated and mock-treated samples using Nexus Expression 3.0 software. Further data analysis was performed using R/Bioconductor and IPA. RESULTS: A total of 1144 genes were regulated. Hierarchical clustering subdivided the groups into two clusters containing the lowest and highest absorbed dose levels, respectively, and revealed similar transcriptional regulation patterns for many kallikrein-related genes. Twenty-seven of the 1144 genes were recurrently regulated after (131)I and (211)At exposure and divided into six clusters. Several signalling pathways were affected, including calcium, integrin-linked kinase, and thyroid cancer signalling, and the peroxisomal proliferator-activated receptor network. CONCLUSIONS: Substantial changes in transcriptional regulation were shown in (131)I and (211)At-treated samples, and 27 genes were identified as potential biomarkers for (131)I and (211)At exposure. Clustering revealed distinct differences between transcriptional profiles of both similar and different exposures, demonstrating the necessity for better understanding of radiation-induced effects on cellular activity. Additionally, ionizing radiation-induced changes in kallikrein gene expression and identified canonical pathways should be further assessed.

Transcriptional Response in Mouse Thyroid Tissue after 211At Administration: Effects of Absorbed Dose, Initial Dose-Rate and Time after Administration.

BACKGROUND: 211At-labeled radiopharmaceuticals are potentially useful for tumor therapy. However, a limitation has been the preferential accumulation of released 211At in the thyroid gland, which is a critical organ for such therapy. The aim of this study was to determine the effect of absorbed dose, dose-rate, and time after 211At exposure on genome-wide transcriptional expression in mouse thyroid gland. METHODS: BALB/c mice were i.v. injected with 1.7, 7.5 or 100 kBq 211At. Animals injected with 1.7 kBq were killed after 1, 6, or 168 h with mean thyroid absorbed doses of 0.023, 0.32, and 1.8 Gy, respectively. Animals injected with 7.5 and 100 kBq were killed after 6 and 1 h, respectively; mean thyroid absorbed dose was 1.4 Gy. Total RNA was extracted from pooled thyroids and the Illumina RNA microarray platform was used to determine mRNA levels. Differentially expressed transcripts and enriched GO terms were determined with adjusted p-value <0.01 and fold change >1.5, and p-value <0.05, respectively. RESULTS: In total, 1232 differentially expressed transcripts were detected after 211At administration, demonstrating a profound effect on gene regulation. The number of regulated transcripts increased with higher initial dose-rate/absorbed dose at 1 or 6 h. However, the number of regulated transcripts decreased with mean absorbed dose/time after 1.7 kBq 211At administration. Furthermore, similar regulation profiles were seen for groups administered 1.7 kBq. Interestingly, few previously proposed radiation responsive genes were detected in the present study. Regulation of immunological processes were prevalent at 1, 6, and 168 h after 1.7 kBq administration (0.023, 0.32, 1.8 Gy).

Transcriptional response in normal mouse tissues after i.v. (211)At administration - response related to absorbed dose, dose rate, and time.

BACKGROUND: In cancer radiotherapy, knowledge of normal tissue responses and toxicity risks is essential in order to deliver the highest possible absorbed dose to the tumor while maintaining normal tissue exposure at non-critical levels. However, few studies have investigated normal tissue responses in vivo after (211)At administration. In order to identify molecular biomarkers of ionizing radiation exposure, we investigated genome-wide transcriptional responses to (very) low mean absorbed doses from (211)At in normal mouse tissues. METHODS: Female BALB/c nude mice were intravenously injected with 1.7 kBq (211)At and killed after 1 h, 6 h, or 7 days or injected with 105 or 7.5 kBq and killed after 1 and 6 h, respectively. Controls were mock-treated. Total RNA was extracted from tissue samples of kidney cortex and medulla, liver, lungs, and spleen and subjected to microarray analysis. Enriched biological processes were categorized after cellular function based on Gene Ontology terms. RESULTS: Responses were tissue-specific with regard to the number of significantly regulated transcripts and associated cellular function. Dose rate effects on transcript regulation were observed with both direct and inverse trends. In several tissues, Angptl4, Per1 and Per2, and Tsc22d3 showed consistent transcript regulation at all exposure conditions. CONCLUSIONS: This study demonstrated tissue-specific transcriptional responses and distinct dose rate effects after (211)At administration. Transcript regulation of individual genes, as well as cellular responses inferred from enriched transcript data, may serve as biomarkers in vivo. These findings expand the knowledge base on normal tissue responses and may help to evaluate and limit side effects of radionuclide therapy.