Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation

Abstract

Recent findings in populations exposed to ionizing radiation (IR) indicate dose-related lens opacification occurs at much lower doses (<2 Gy) than indicated in radiation protection guidelines. As a result, research efforts are now being directed towards identifying early predictors of lens degeneration resulting in cataractogenesis. In this study, Raman micro-spectroscopy was used to investigate the effects of varying doses of radiation, ranging from 0.01 Gy to 5 Gy, on human lens epithelial (HLE) cells which were chemically fixed 24 h post-irradiation. Raman spectra were acquired from the nucleus and cytoplasm of the HLE cells. Spectra were collected from points in a 3 × 3 grid pattern and then averaged. The raw spectra were preprocessed and principal component analysis followed by linear discriminant analysis was used to discriminate between dose and control for 0.25, 0.5, 2, and 5 Gy. Using leave-one-out cross-validation accuracies of greater than 74% were attained for each dose/control combination. The ultra-low doses 0.01 and 0.05 Gy were included in an analysis of band intensities for Raman bands found to be significant in the linear discrimination, and an induced repair model survival curve was fit to a band-difference-ratio plot of this data, suggesting HLE cells undergo a nonlinear response to low-doses of IR. A survival curve was also fit to clonogenic assay data done on the irradiated HLE cells, showing a similar nonlinear response.

Computational Cell Dosimetry for Cancer Radiotherapy and Diagnostic Radiology

“…Building on these general observations based on MC results from part 1, estimates of microdosimetric spread for some example studies from the recent literature may be obtained using equation 6.2 with fit parameters from part 2 (table 6.1). Allen et al 70 used RS sampling volumes equivalent to the small sampling regions considered herein, irradiating HLE cells using a 120 kVp x-ray spectrum and considering doses between 0.01 and 5 Gy. In this case, the microdosimetric spread is 403% at 0.01 Gy, decreasing to 18% at 5 Gy.…”

“…the small sampling volumes are arranged in a 3 × 3 grid in the xy-plane, with 3 μm separating the centres of adjacent sampling volumes (as in Allen et al 70 ). Only a single large sampling volume is simulated at the centre of each nucleus ( figure 6.1(b)).…”

“…Furthermore, recent studies have used Raman spectroscopy to probe cell response to radiation using μm-sized sampling volumes, and considering radiation doses < 1 Gy. 69,70 Raman spectroscopy can be used to assess radiosensitivity, as well as investigate radiation-induced damage to particular subcellular components based on knowledge of molecular vibration frequencies.…”

“…Maguire et al69 demonstrated that RS is capable of detecting 60 Co radiation response in human lymphocytes for doses as low as 0.05 Gy; DNA damage predicted by changes in Raman spectra were confirmed by comparison with γ-H2AX assay results. Allen et al70 irradiated human lens epithelial (HLE) cells with 120 kVp x-rays to doses as low as 0.01 Gy, finding that Raman band intensities for various proteins and nucleic acids in the nucleus exhibit non-linear behaviour as a function of dose below 0.5 Gy; they noted that the corresponding cell survival curve demonstrates a similar shape (indicative of a region of low dose hypersensitivity followed by a region of radio-resistance). Meade et al146 used RS to observe the same dose response trends in human keratinocyte (HaCaT) cells exposed to60 Co radiation, considering doses as low as 0.005 Gy (in addition to investigations of the bystander effect).Despite the recent progress in applying RS to probe radiation response on microscopic (cellular) levels, studies have relied on the macroscopic dose to quantify the amount of energy deposited without considering the stochastic nature of radiation transport and energy deposition.…”

“…The minimum cell separation distance is 2.06 μm 35. Two sizes of sampling volumes within the nucleus are considered: a 'small' cylinder with 1 μm diameter and 4 μm height,70 and a 'large' rectangular prism measuring 2 × 5 × 10 μm 3 . 140…”