Role of blood derived cell fractions, temperature and sample transport on gene expression-based biological dosimetry

Nasser, Farah; Cruz-García, Lourdes; O’Brien, Gráinne; Badie, Christophe

doi:10.1080/09553002.2021.1906464

Cited by 4 publications

(4 citation statements)

References 59 publications

(91 reference statements)

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“…It is important to keep in mind that the relationships described here are valid for different fractions of circulating leukocytes (Nasser et al 2021). For other cells, regulation of FDXR expression after irradiation may look slightly different and therefore conclusions of this study should not be transferred to other cellular systems too hastily.…”

Section: Discussionmentioning

confidence: 93%

Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry – premises and pitfalls

Brzóska,

Abend,

O’Brien

et al. 2024

International Journal of Radiation Biology

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Radiation-induced alterations in gene expression show great promise for dose reconstruction and for severity prediction of acute health effects. Among several genes explored as potential biomarkers, FDXR is widely used due to high upregulation in white blood cells following radiation exposure. Nonetheless, the absence of a standardized protocols for gene expression-based biodosimetry is a notable gap that warrants attention to enhance the accuracy, reproducibility and reliability of such methods. The primary objective of this study was to evaluate the sensitivity of transcriptional biodosimetry to differences in protocols used by different laboratories and establish guidelines for the calculation of calibration curve using FDXR expression data. Additionally, we aimed to investigate the feasibility of extrapolating a calibration curve derived from samples irradiated with doses exceeding 0.1 Gy to doses below this threshold. Material and MethodsTwo sets of irradiated blood samples generated during RENEB exercise were used.The first included samples irradiated with known doses including: 0, 0.25, 0.5, 1, 2, 3 and 4 Gy. The second set consisted of three 'blind' samples irradiated with 1.8 Gy, 0.4 Gy and a sham-irradiated sample. After irradiation, samples were incubated at 37 °C over 24 h. Subsequently, aliquots were filled into PAXgene tubes and sent to participating laboratories where RNA isolation and FDXR expression analysis by qPCR were performed. ResultsCalibration curves were generated using non-linear and linear regression. Dose estimates for nonirradiated sample and sample irradiated with 0.4 Gy showed remarkable consistency across all laboratories, closely approximating the true doses regardless methodological variations. For sample irradiated with 1.8 Gy the differences were more pronounced but remained within an acceptable margin for triage within the context of high dose range. ConclusionMethodological differences in reference genes and primers/probes used for FDXR expression measurement do not have a significant impact on the dose estimates generated, provided that all reference genes performed as expected and the primers/probes target a similar set of transcript variants. The preferred method for constructing a calibration curve based on FDXR expression data involves employing linear regression to establish a function that describes the relationship between the logarithm of absorbed dose and FDXR Ct values. However, one should be careful with using non-irradiated sample data as these cannot be accurately represented on a logarithmic scale. A standard curve generated using this approach can give reliable dose estimations in a dose range from 50 mGy to 4 Gy at least.

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Section: Discussionmentioning

confidence: 93%

Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry – premises and pitfalls

Brzóska,

Abend,

O’Brien

et al. 2024

International Journal of Radiation Biology

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“…Peripheral blood was used as a test sample, as it is easy to collect by a less invasive method; also, even a small quantity can be a representative sample of the entire body. The above genes were selected as they are reported to mediate typical cellular response upon radiation exposure (checkpoint activation, repair, and apoptosis) and established a role in the DNA damage response [Abend et al, 2016;Knops et al, 2012;Manning et al, 2013Manning et al, , 2017Nasser et al, 2021;Paul and Amundson, 2008]. An interindividual variation in the levels of that gene signature is observed among the healthy participants as the normalized ΔCt values ranged from 5.…”

Section: Discussionmentioning

confidence: 99%

“…This raises the concern that inflammation affects radiation responses and may impact the use of genes such as FDXR in radiation triage and biodosimetry considering ionizing radiation is a wellknown pro-inflammatory agent [Mukherjee et al, 2019]. In addition, sample processing [Nasser et al, 2021], gender [Li et al, 2019;Manning et al, 2013], lifestyle [Cruz-Garcia et al, 2018;Manning et al, 2017], the genetic background of an individual, and pre-existing disease conditions [Agbenyegah et al, 2018;Ostheim et al, 2022] can significantly affect the response to radiation exposure. Furthermore, these studies cautioned that exposure to ionizing radiation induces multiple signal transduction pathways; a single gene expression change is unlikely to represent a specific response to ionizing radiation.…”

Section: Introductionmentioning

confidence: 99%

Candidate Gene Expression in Regional Population and Its Relevance for Radiation Triage

Kannan¹,

Koshy²,

Raavi³

et al. 2023

Cytogenet Genome Res

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Quantification of gene expression signatures has been substantiated as a potential and rapid marker for radiation triage and biodosimetry during nuclear emergencies. Similar to the established biodosimetry assays, the gene expression assay has drawbacks such as being highly dynamic and transient, not specific to ionizing radiation, and also influenced by confounding factors such as gender, health status, lifestyle, and inflammation. In view of that, prior knowledge of baseline expression of certain candidate genes in a population could complement the discrimination of the unexposed from the exposed individuals without the need for individual pre-exposure controls. We intended to establish a baseline expression of reported radiation-responsive genes such as CDKN1A, DDB2, FDXR, and PCNA in the blood samples of healthy human participants and then compare it with diabetic/hypertension participants (as a chronic inflammatory condition) drawn from south Indian population. Further, we have examined the appropriateness of the assay for radiation triage-like situations; i.e., the expression profiles of those genes were examined in the participants who underwent X-ray-based medical imaging. Acute inflammation induced by lipopolysaccharide exposure in the blood significantly increased the fold expression of those genes (p < 0.0001) compared to the control. Whereas the basal expression level of those genes among the participants with the inflammatory condition is marginally higher than those observed in the healthy participants; despite the excess, the fold increase in those genes between the groups did not differ significantly. Consistent with the inflammatory participants, the basal expression level of those genes in the blood sample of participants who received X-radiation during neuro-interventional and computed tomography imaging is marginally higher than those observed in the pre-exposure of respective groups. Nevertheless, the fold increase in those genes did not differ significantly as the fold change fell within the two folds. Thus, overall results suggest that the utility of CDKN1A, DDB2, FDXR, and PCNA gene expression for radiation triage specific after very low-dose radiation exposure needs to be interpreted with caution for a much more reliable triage.

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“…It should be noted that we used isolated PBMCs for in vitro studies while all leukocytes (PBMCs plus granulocytes) were lysed in PAXGene tubes. However, no significant differences in FDXR expression were found between WBC and PBMCs at 24 h following a 2 Gy dose [ 88 ].…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Dynamics of DNA Damage Responsive Genes in Circulating Leukocytes during Radiotherapy

Cruz-García¹,

Nasser²,

O’Brien³

et al. 2022

Cancers

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External beam radiation therapy leads to cellular activation of the DNA damage response (DDR). DNA double-strand breaks (DSBs) activate the ATM/CHEK2/p53 pathway, inducing the transcription of stress genes. The dynamic nature of this transcriptional response has not been directly observed in vivo in humans. In this study we monitored the messenger RNA transcript abundances of nine DNA damage-responsive genes (CDKN1A, GADD45, CCNG1, FDXR, DDB2, MDM2, PHPT1, SESN1, and PUMA), eight of them regulated by p53 in circulating blood leukocytes at different time points (2, 6–8, 16–18, and 24 h) in cancer patients (lung, neck, brain, and pelvis) undergoing radiotherapy. We discovered that, although the calculated mean physical dose to the blood was very low (0.038–0.169 Gy), an upregulation of Ferredoxin reductase (FDXR) gene transcription was detectable 2 h after exposure and was dose dependent from the lowest irradiated percentage of the body (3.5% whole brain) to the highest, (up to 19.4%, pelvic zone) reaching a peak at 6–8 h. The radiation response of the other genes was not strong enough after such low doses to provide meaningful information. Following multiple fractions, the expression level increased further and was still significantly up-regulated by the end of the treatment. Moreover, we compared FDXR transcriptional responses to ionizing radiation (IR) in vivo with healthy donors’ blood cells exposed ex vivo and found a good correlation in the kinetics of expression from the 8-hours time-point onward, suggesting that a molecular transcriptional regulation mechanism yet to be identified is involved. To conclude, we provided the first in vivo human report of IR-induced gene transcription temporal response of a panel of p53-dependant genes. FDXR was demonstrated to be the most responsive gene, able to reliably inform on the low doses following partial body irradiation of the patients, and providing an expression pattern corresponding to the % of body exposed. An extended study would provide individual biological dosimetry information and may reveal inter-individual variability to predict radiotherapy-associated adverse health outcomes.

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Role of blood derived cell fractions, temperature and sample transport on gene expression-based biological dosimetry

Cited by 4 publications

References 59 publications

Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry – premises and pitfalls

Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry – premises and pitfalls

Candidate Gene Expression in Regional Population and Its Relevance for Radiation Triage

Transcriptional Dynamics of DNA Damage Responsive Genes in Circulating Leukocytes during Radiotherapy

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