Construction of dose response curve for 6 MV LINAC X-rays using Premature Chromosome Condensation assay for radiation dosimetry

Highlights

• This study is first of its kind to construct dose response curve for LINAC X-rays using PCC assay.

• Chemically induced PCC is very reliable biodosimetry tool for accidental exposure.

• PCC rings observed to be appropriate biomarker for high dose exposure.

• Different types of PCC rings observed in various phases of cell cycle (G2/M, M, M/A) are shown.

• It is shown that the dicentric chromosomes are suitable only for low dose exposure (<5 Gy).

Abstract

Quantification of chromosomal aberrations in the exposed personnel blood samples is considered as a ‘gold standard’ and sensitive biomarker in biological dosimetry. Despite technological developments, culture of cells for 48–52 h remains an unmet need in case of triage biodosimetry. Moreover, it is difficult to get sufficient number of metaphase spreads for scoring after high doses of exposures. The technique which causes condensation of chromatin before mitosis using biological or chemical agent is named as Premature Chromosome Condensation (PCC) assay. This assay is considered as an alternative to chromosome aberration assay, particularly at high acute doses of low and high LET radiation. To establish the PCC assay, blood samples were collected from healthy non-smoking individuals (n = 3) and exposed to various doses (0–20 Gy) of 6 MV X-rays at a dose rate of 5.6 Gy/min, using a high energy Linear accelerator (LINAC). Irradiated blood samples were subjected to Calyculin-A induced PCC. About 500 cells or more than 100 Ring Chromosomes (RC) were scored at each dose. Dicentric chromosomes (DC) and acentric fragments were also scored at each dose; the number of chromosomal aberrations in G1, M, G2/M and M/A phase of cell cycle were recorded and the frequency was used to construct the dose response curve. A dose dependent increase in RC and DC frequency were observed with a slope of 0.049 ± 0.002 and 0.30 ± 0.02 respectively. This study is first of its kind to construct a dose response curve for LINAC X-rays using a PCC assay.

Introduction

A radiation biomarker is a biological entity that alters after radiation exposure. Biomarkers are mainly required to estimate the received dose, to detect the radiation induced health effects and to investigate the radiation susceptibility of individuals. During mass radiation casualties, proper medical treatment of exposed individuals is required as quickly as possible based on dose estimates provided by biodosimetry techniques. There are various cytogenetic biomarkers such as dicentric chromosomes (DC), micronuclei (MN), translocation, gamma-H2AX foci and ring chromosomes (RC). Among these biomarkers, DC, MN, gamma-H2AX foci and RC provide the dose estimate of recent radiation exposure whereas translocation using Fluorescence in situ hybridization (FISH) technique provides the dose estimate of past radiation exposure.

Scoring of DC from peripheral blood lymphocytes (PBL) at first mitotic cell division are considered as ‘gold standard’, highly sensitive and reliable method to estimate the dose received by individuals (IAEA, 2011). But the scoring of MN is easier and faster than that of DC for dose assessment (Blakely, et al., 2001). However, the DC assay and Cytokinesis Blocked Micronucleus (CBMN) assay can be reliably used for dose estimation only when the exposure is up to 5 Gy (IAEA, 2011; Vral et al., 2011; Goransson Nyberg et al, 2011Goransson Nyberg et al, 2011; Swartz, 2014). When the irradiation dose increases (>5 Gy), then the damaged cells stop cell cycling in G1/G2 phase and the cells do not enter into mitosis. Hence, obtaining the metaphase cells after high dose exposure is extremely difficult (Gotoh, 2015). For doses above 5 Gy, the dose response of these assays shows sign of saturation due to mitotic delay and cell death (Puig, 2013; Karachristou, 2015).

Premature Chromosome Condensation (PCC) assay is considered as an important assay for biodosimetry in cases of accidental/high dose radiation exposure owing to following reasons: (i) This assay causes chromosome condensation before first mitosis (ii) simple and reliable procedure which involves scoring of rings in Giemsa stained chromosomes. (iii) Rings can be scored at any phase of cell cycle, hence no sign of saturation at higher dose. Moreover, this assay has shown to be the most suitable assay for estimating acute, chronic whole and partial body exposure (Balakrishnan, 2010; Puig, 2013, Gotoh, 2015).

PCC can be induced using chemical compounds such as Calyculin A or Okadaic acid (Prasanna et al., 2000; Hosseini, 2004). By using this technique every phase of cell cycle such as G1, G2/M, M and M/A can be discriminated with reasonably high PCC index. There are various published reports for the dose response calibration curves for low LET radiation (‘Co-60’ gamma rays) using the PCC assay (Lamadrid et al., 2007; Balakrishnan et al., 2010; Lindholm et al., 2010; Puig et al., 2013). However, we do not come across any publications for the dose response calibration curve for high energy 6 MV Linear accelerator (LINAC) X-rays using PCC assay.

The first step towards the adoptability of any assay for the biodosimetry is to establish a reference calibration curve. The International Atomic Energy Agency (IAEA) has recommended that each cytogenetic biodosimetry laboratory should possess own reference calibration curve (IAEA, 2011). The Atomic Energy Regulatory Board (AERB), India, accredited biodosimetry laboratory of Sri Ramachandra Institute of Higher Education and Research has already established the dose-response curves for DC, MN and gamma-H2AX assay for the dosimetry purpose. To share the workload during emergency, the established assay has been validated by inter-laboratory comparison exercise among various laboratories (Manivannan et al, 2014; Tamizhselvan et al., 2014; Venkateswarlu et al., 2015). To quantify the dose using DC and MN assay require proliferating cells; whereas in gamma-H2AX, either the intensity reaches saturation at higher doses or frequency is reduced in delayed sampling. During radiological emergency, it is essential to assess the dose received by an individual exposed to very high doses as early as possible in order to proceed with effective medical management. In view of the above, we have standardized the chemically induced PCC and constructed a dose-response curve for the X-rays (6 MV LINAC at 5.6 Gy/min) in the dose range of 0–20 Gy.