Inter-Individual Differences in Radiosensitivity Based on Micronucleus assay and CDKN1A mRNA: A Preliminary Report

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Introduction

Exposure to ionizing radiation induces measurable cellular damage that is proportional to the dose. This is the basis for extrapolation of absorbed dose from an established dose-response curve in cytogenetic biodosimetry, with the premise that radiation response is uniform in a certain population. However, the issue of inter-individual differences in radiosensitivity exists because of inherent genetic mutations that influence radiation response. We therefore examined if this variation in radiosensitivity is detected at the cellular level using cytokinesis blocked micronucleus assay. We further studied variation at the molecular level by quantifying expression of radiation-inducible transcripts for cell cycle arrest (CDKN1A) and growth arrest (GADD45A) as these were previously associated with radiosensitivity.

Materials and Methods

Blood samples were collected from healthy donors and exposed in vitro to 2 Gy of gamma radiation. At 1 hour after irradiation, 0.5 mL of blood was incubated at 37°C in 5% CO₂ for 72 h. At the end of incubation, micronuclei (MN) were scored and its frequency in binucleated cells were used to estimate the absorbed dose from the previously established dose-response curve. For gene expression, lymphocytes from 1 ml blood were isolated and RNA was extracted from 3x10⁶ cells. To measure transcription levels, reverse transcription was carried out on 250 ng RNA followed by quantitative real-time PCR using SYBR Green method.

Results and Discussion

Micronuclei scoring of non-irradiated blood samples revealed baseline MN frequencies ranging from 0 to 0.009. After 2 Gy exposure, micronuclei frequency in all samples increased but in varying degrees between 0.009 and 0.06. Absorbed dose estimation indicated that samples 1 and 6, which have the highest MN frequency of 0.06 and 0.05, received absorbed doses of 2.99 Gy (95% C.I. 2.45-3.61) and 2.80 Gy (95% C.I. 1.67-4.19), respectively. This suggests that despite exposure to the same dose, magnitude of MN induction may vary from person to person which are reflected in the absorbed dose estimates. Radiation likewise increased the transcription of both CDKN1A and GADD45A in 100% of samples but only CDKN1A transcription positively correlated with MN frequency (R2 = 0.87, p<0.05). CDKN1A transcription increased by more than 11-fold and 5-fold in samples 1 and 6, respectively, whereas the less radiosensitive samples increased by only one to two-fold. Past studies have shown similar correlation with inherent radiosensitivity (Zyla et al, 2019; Alsbeih et al., 2007) and acute tissue reactions to radiotherapy (Badie et al., 2008). It may be because CDKN1A or p21 is one of the genes involved in micronuclei formation (Leeuwen et al., 2011). It functions by inhibiting cell cycle progression upon stimulation by stress, including DNA damage, until such is repaired. Hence, CDKN1A might be a reliable, but not specific, indicator of radiosensitivity in conjunction with MN assay. Future studies on a larger sample size with transcripts more specific to ionizing radiation are recommended.