B1 Sequence–Based Real-Time Quantitative PCR: A Sensitive Method for Direct Measurement of Mouse Plasma DNA Levels After Gamma Irradiation

Abstract

Purpose-Current biodosimetric techniques for determining radiation exposure have inherent delays, and quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA.Methods and Materials-Real-time quantitative PCR was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 hrs from mice receiving 0-10 Gy total-or partial-body irradiation [ 137 Cs γ-ray source at ≈1.86 Gy/min (homogeneity: ±6.5%)].Results-The correlation coefficient between DNA levels and the threshold cycle value (C T ) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy TBI, plasma DNA levels gradually increased beginning at 3 hours after irradiation, peaked at 9 hours, and returned to baseline within 48 hours. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 hours after collection of plasma samples.Conclusions-A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2-10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

PicoGreen Assay of Circular DNA for Radiation Biodosimetry

“…Using a less sensitive assay, Belokhvostov et al found that plasma DNA levels were altered after high-dose irradiation in a rat model (28). Comparing our results using PicoGreen with previously published methods (PCR of the B1 gene sequence not shown here) demonstrates the strong correlation between the data shown here (for PicoGreen) and PCR of the B1 gene sequence; further, our data show a time and dose dependence that peaked at 9 h postirradiation, that provides a dose response in the clinically relevant range of 2–10 Gy (27). The potential of the PicoGreen assay as a biodosimeter is further supported by experiments performed in additional mouse strains of different backgrounds (Figs.…”

“…Other types of biodosimetry methods include: radiation-induced damage and repair of DNA (15–17); the consequences of misrepair (4, 18–21) including micronuclei production (4, 18); gene expression profiles, as determined by microarrays (19, 20) and reverse transcription polymerase chain reactions (RT-PCR) (21); and the measurement of dsDNA released into plasma as a consequence of radiation-induced cell death, which is influenced by apoptotic and nonapoptotic mechanisms (21–28). Our group has previously shown that inter-B1 sequences can be used as a trace marker to measure the circulating dsDNA by real-time quantitative PCR in irradiated mice and is a potentially useful biodosimeter for radiation and other combined toxicities (27). However, this method requires not only restricted environmental conditions to prevent contamination of the samples but also operation by trained personnel, limiting its application for very large events.…”

Increased circulating cell‐free DNA levels and mtDNA fragments in interventional cardiologists occupationally exposed to low levels of ionizing radiation

“…To date, little is known about changes in ccf‐DNA concentrations after radiation exposure [Leon et al, ; Zhang et al, ; Cheng et al, ]. Radiation‐released circulating ccf‐DNA has been actively studied in experimental models after exposure to high [Vladimirov et al, ; Vasilyeva et al, , ] and low doses [Shishkina et al, ].…”

“…To date, little is known about changes in ccf-DNA concentrations after radiation exposure [Leon et al, 1977;Zhang et al, 2009;Cheng et al, 2009]. Radiation- Environmental and Molecular Mutagenesis.…”

“…Ionizing radiation induces a relatively high incidence of mtDNA mutation, deletion, or depletion and is likely to trigger programmed cell death and the release of mtDNA [Murphy et al, ; Wilding et al, ]. To date, several studies demonstrated changes in ccf‐DNA and ccf‐mtDNA concentrations in response to high radiation exposure [Leon et al, ; Cheng et al, ; Abdullaev et al, ; Zhang et al, ], but the impact of occupational radiation exposure is still lacking. The purpose of this study was to investigate the impact of chronic low‐dose radiation exposure on serum ccf‐DNA and ccf‐mtDNA of interventional cardiologists working in high‐volume Cath Lab in order to assess its possible role as a new useful radiation biomarker.…”