Gene Expression-Based Detection of Radiation Exposure in Mice after Treatment with Granulocyte Colony-Stimulating Factor and Lipopolysaccharide

Abstract

In a large-scale nuclear incident, many thousands of people may be exposed to a wide range of radiation doses. Rapid biological dosimetry will be required on an individualized basis to estimate the exposures and to make treatment decisions. To ameliorate the adverse effects of exposure, victims may be treated with one or more cytokine growth factors, including granulocyte colony-stimulating factor (G-CSF), which has therapeutic efficacy for treating radiation-induced bone marrow ablation by stimulating granulopoiesis. The existence of infections and the administration of G-CSF each may confound the ability to achieve reliable dosimetry by gene expression analysis. In this study, C57BL/6 mice were used to determine the extent to which G-CSF and lipopolysaccharide (LPS, which simulates infection by gram-negative bacteria) alter the expression of genes that are either radiation-responsive or non-responsive, i.e., show potential for use as endogenous controls. Mice were acutely exposed to (60)Co γ rays at either 0 Gy or 6 Gy. Two hours later the animals were injected with either 0.1 mg/kg of G-CSF or 0.3 mg/kg of LPS. Expression levels of 96 different gene targets were evaluated in peripheral blood after an additional 4 or 24 h using real-time quantitative PCR. The results indicate that the expression levels of some genes are altered by LPS, but altered expression after G-CSF treatment was generally not observed. The expression levels of many genes therefore retain utility for biological dosimetry or as endogenous controls. These data suggest that PCR-based quantitative gene expression analyses may have utility in radiation biodosimetry in humans even in the presence of an infection or after treatment with G-CSF.

DNA Repair and Cell Cycle Biomarkers of Radiation Exposure and Inflammation Stress in Human Blood

“…Expression of GADD45a, LIG1 and XPC were decreased at 24 hours after 6 Gy IR in mice, whereas we observed increased expression at 24 hrs after 2 Gy in our ex vivo human blood culture model consistent with published human ex vivo and in vivo literature [7], [12], [30]. Also, our use of a 2 Gy exposure (rather than 6 Gy used in a prior mouse study [30]) is more relevant for radiation biodosimetry because individuals having a radiation exposure dosage of less than 2 Gy require no immediate treatment as opposed to those having a dosage higher than 2 Gy. The inherent differences between murine and human assays emphasize the importance of using human model systems to validate biomarkers for human radiation biodosimetry.…”

“…However, in that study DDB2 was downregulated and no significant changes were observed for FDXR or XPC, which is inconsistent with our results and those of others in humans irradiated ex vivo [10]. Expression of GADD45a, LIG1 and XPC were decreased at 24 hours after 6 Gy IR in mice, whereas we observed increased expression at 24 hrs after 2 Gy in our ex vivo human blood culture model consistent with published human ex vivo and in vivo literature [7], [12], [30]. Also, our use of a 2 Gy exposure (rather than 6 Gy used in a prior mouse study [30]) is more relevant for radiation biodosimetry because individuals having a radiation exposure dosage of less than 2 Gy require no immediate treatment as opposed to those having a dosage higher than 2 Gy.…”

“…Our finding seriously undermines the promise of CDKN1A as a predictive tool for radiation exposure in individuals suffering simultaneous inflammatory stress. Studies in the murine central nervous system also identified CDKN1A as an inflammatory response gene [44] and LPS exposure upregulated CDKN1A transcripts in mice [30]. LPS-induced and the radiation-induced CDKN1A responses were indistinguishable in our human blood model, while in the mouse the upregulation of CDKN1A at 24 hours after LPS injection did not mask the ability to detect a radiation response [30].…”

“…Studies in the murine central nervous system also identified CDKN1A as an inflammatory response gene [44] and LPS exposure upregulated CDKN1A transcripts in mice [30]. LPS-induced and the radiation-induced CDKN1A responses were indistinguishable in our human blood model, while in the mouse the upregulation of CDKN1A at 24 hours after LPS injection did not mask the ability to detect a radiation response [30]. This difference in murine vs human responses might be attributed to the differences in LPS dosage (50 ng/ml in our study vs. 0.3 mg/kg which equals 7.2 µg per mouse), LPS bioavailability and species differences in response.…”

“…IR exposure is known to modulate transcript and/or protein levels of several cell cycle regulators ( CDKN1A ( p21 ), GADD45a, Cyclin G1 ( CCNG1 ), CHK2 -thr68) and apoptosis genes ( BAX and BBC3) in diverse cell and blood model systems ( in vivo , in vitro and ex vivo) [6], [8], [10], [23]–[28]. However, little is known of how co-exposure to confounding factors can affect the utility of individual biomarkers for radiation biodosimetry [1], [29], [30].…”