Applicability of Gene Expression in Saliva as an Alternative to Blood for Biodosimetry and Prediction of Radiation-induced Health Effects.

As the great majority of gene expression (GE) biodosimetry studies have been performed using blood as the preferred source of tissue, searching for simple and less-invasive sampling methods is important when considering biodosimetry approaches. Knowing that whole saliva contains an ultrafiltrate of blood and white blood cells, it is expected that the findings in blood can also be found in saliva. This human in vivo study aims to examine radiation-induced GE changes in saliva for biodosimetry purposes and to predict radiation-induced disease, which is yet poorly characterized. Furthermore, we examined whether transcriptional biomarkers in blood can also be found equivalently in saliva. Saliva and blood samples were collected in parallel from radiotherapy (RT) treated patients who suffered from head and neck cancer (n = 8) undergoing fractioned partial-body irradiations (1.8 Gy/fraction and 50-70 Gy total dose). Samples were taken 12-24 h before first irradiation and ideally 24 and 48 h, as well as 5 weeks after radiotherapy onset. Due to the low quality and quantity of isolated RNA samples from one patient, they had to be excluded from further analysis, leaving a total of 24 saliva and 24 blood samples from 7 patients eligible for analysis. Using qRT-PCR, 18S rRNA and 16S rRNA (the ratio being a surrogate for the relative human RNA/bacterial burden), four housekeeping genes and nine mRNAs previously identified as radiation responsive in blood-based studies were detected. Significant GE associations with absorbed dose were found for five genes and after the 2nd radiotherapy fraction, shown by, e.g., the increase of CDKN1A (2.0 fold, P = 0.017) and FDXR (1.9 fold increased, P = 0.002). After the 25th radiotherapy fraction, however, all four genes (FDXR, DDB2, POU2AF1, WNT3) predicting ARS (acute radiation syndrome) severity, as well as further genes (including CCNG1 [median-fold change (FC) = 0.3, P = 0.013], and GADD45A (median-FC = 0.3, P = 0.031)) appeared significantly downregulated (FC = 0.3, P = 0.01-0.03). A significant association of CCNG1, POU2AF1, HPRT1, and WNT3 (P = 0.006-0.04) with acute or late radiotoxicity could be shown before the onset of these clinical outcomes. In an established set of four genes predicting acute health effects in blood, the response in saliva samples was similar to the expected up- (FDXR, DDB2) or downregulation (POU2AF1, WNT3) in blood for up to 71% of the measurements. Comparing GE responses (PHPT1, CCNG1, CDKN1A, GADD45A, SESN1) in saliva and blood samples, there was a significant linear association between saliva and blood response of CDKN1A (R2 = 0.60, P = 0.0004). However, the GE pattern of other genes differed between saliva and blood. In summary, the current human in vivo study, (I) reveals significant radiation-induced GE associations of five transcriptional biomarkers in salivary samples, (II) suggests genes predicting diverse clinical outcomes such as acute and late radiotoxicity as well as ARS severity, and (III) supports the view that blood-based GE response can be reflected in saliva samples, indicating that saliva is a "mirror of the body" for certain but not all genes and, thus, studies for each gene of interest in blood are required for saliva.

RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay.

Early and high-throughput individual dose estimates are essential following large-scale radiation exposure events. In the context of the Running the European Network for Biodosimetry and Physical Dosimetry (RENEB) 2021 exercise, gene expression assays were conducted and their corresponding performance for dose-assessment is presented in this publication. Three blinded, coded whole blood samples from healthy donors were exposed to 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min) using the X-ray source Yxlon. These exposures correspond to clinically relevant groups of unexposed, low dose (no severe acute health effects expected) and high dose exposed individuals (requiring early intensive medical health care). Samples were sent to eight teams for dose estimation and identification of clinically relevant groups. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray analyses, samples were lysed, stored at 20°C and shipped on wet ice. RNA isolations and assays were run in each laboratory according to locally established protocols. The time-to-result for both rough early and more precise later reports has been documented where possible. Accuracy of dose estimates was calculated as the difference between estimated and reference doses for all doses (summed absolute difference, SAD) and by determining the number of correctly reported dose estimates that were defined as ±0.5 Gy for reference doses <2.5 Gy and ±1.0 Gy for reference doses >3 Gy, as recommended for triage dosimetry. We also examined the allocation of dose estimates to clinically/diagnostically relevant exposure groups. Altogether, 105 dose estimates were reported by the eight teams, and the earliest report times on dose categories and estimates were 5 h and 9 h, respectively. The coefficient of variation for 85% of all 436 qRT-PCR measurements did not exceed 10%. One team reported dose estimates that systematically deviated several-fold from reported dose estimates, and these outliers were excluded from further analysis. Teams employing a combination of several genes generated about two-times lower median SADs (0.8 Gy) compared to dose estimates based on single genes only (1.7 Gy). When considering the uncertainty intervals for triage dosimetry, dose estimates of all teams together were correctly reported in 100% of the 0 Gy, 50% of the 1.2 Gy and 50% of the 3.5 Gy exposed samples. The order of dose estimates (from lowest to highest) corresponding to three dose categories (unexposed, low dose and highest exposure) were correctly reported by all teams and all chosen genes or gene combinations. Furthermore, if teams reported no exposure or an exposure >3.5 Gy, it was always correctly allocated to the unexposed and the highly exposed group, while low exposed (1.2 Gy) samples sometimes could not be discriminated from highly (3.5 Gy) exposed samples. All teams used FDXR and 78.1% of correct dose estimates used FDXR as one of the predictors. Still, the accuracy of reported dose estimates based on FDXR differed considerably among teams with one team's SAD (0.5 Gy) being comparable to the dose accuracy employing a combination of genes. Using the workflow of this reference team, we performed additional experiments after the exercise on residual RNA and cDNA sent by six teams to the reference team. All samples were processed similarly with the intention to improve the accuracy of dose estimates when employing the same workflow. Re-evaluated dose estimates improved for half of the samples and worsened for the others. In conclusion, this inter-laboratory comparison exercise enabled (1) identification of technical problems and corrections in preparations for future events, (2) confirmed the early and high-throughput capabilities of gene expression, (3) emphasized different biodosimetry approaches using either only FDXR or a gene combination, (4) indicated some improvements in dose estimation with FDXR when employing a similar methodology, which requires further research for the final conclusion and (5) underlined the applicability of gene expression for identification of unexposed and highly exposed samples, supporting medical management in radiological or nuclear scenarios.

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes.

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5-40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5-8 h varying exposure times; second: varying dose rates of 0.5-8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.

Radiation induced COX-2 expression and mutagenesis at non-targeted lung tissues of gpt delta transgenic mice.

BACKGROUND: Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study. METHODS: A 1-cm(2) area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed. RESULTS: Compared with sham-treated controls, the Spi(-) mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR. CONCLUSION: The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis.

Transcript levels of the eukaryotic translation initiation factor 5A gene peak at early G(1) phase of the cell cycle in the dinoflagellate Crypthecodinium cohnii.

A cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate Crypthecodinium cohnii (CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized C. cohnii cells, the transcript level peaked at early G(1) and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (Saccharomyces cerevisiae) and certain mammalian cell types suggest a role for eIF-5A in the G(1)/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while falling within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor D-difluoromethylornithine and the specific hypusination inhibitor N-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinoflagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed.

Occurrence and expression of cspA, a cold shock gene, in Antarctic psychrotrophic bacteria.

The homologue of cold shock gene cspA of Escherichia coli was detected in various isolates of Antarctic psychrotrophs representing both Gram-positive and Gram-negative bacteria. The Northern hybridization study indicated that the transcript size of cspA in the psychrotrophic Gram-positive bacterium Arthrobacter protophormiae and Gram-negative Pseudomonas fluorescens was similar to that of E. coli and that the cspA homologues in these two psychrotrophs were expressed constitutively at a low level both at 4 degrees C and 22 degrees C. In P. fluorescens, the expression of cspA mRNA was inducible after shift of temperature from 22 to 4 degrees C and the maximum level of induction occurred after 1 h which correlated with the time-lag required for growth of the culture after temperature shift.