Countering effects of a combination of podophyllotoxin,podophyllotoxin β-D-glucoside and rutin hydrate in minimizing radiation induced chromosomal damage,ROS and apoptosis in human blood lymphocytes

The present study was conceptualized with the aim of developing a safe radioprotector for human application against radiation induced toxicity. For this study, a formulation (G-002M) prepared by combining three active principles isolated from rhizomes of Podophyllum hexandrum, was evaluated for its potential to protect genomic DNA of human blood cells exposed to different doses of radiation (5,7&10Gy). Blood samples were pretreated (-1hr to exposure) with G-002M. Parameters of Premature Chromosome Condensation (PCC) assay like PCC-index, PCC-rings and PCC-fragments were used to estimate radiation induced chromosomal aberrations. Radiation (7Gy) induce ROS generation and its modulation by G-002M was determined by flow-cytometry and fluorescent microscopy while apoptosis (0,2,24&48 hr) was analyzed using TUNEL assay. Effect on spindle organization in G2/M arrested cells by all the three compounds individually was studied using immunofluorescence microscopy. Irradiation caused dose dependent linear increase in PCC-rings and fragments, while decline in PCC index. G-002M pretreatment significantly decreased these chromosomal aberrations at all the radiation doses and assisted cell survival as indicated by increased PCC index compared with radiation only group. Significant decrease in radiation induced intracellular ROS (45 ± 3%) and apoptosis (49.9%) was also exhibited by the formulation. On podophyllotoxin treatment, most of the cells have shown blocked spindles however, depicted normal arrangement. G-002M also demonstrated a highly significant Dose Modifying Factor or DMF (PCC-rings: 2.27 and PCC-fragments: 1.60). Present study based on many parameters along with DMF study, strongly suggests that G-002M is an effective formulation with a potential to minimize chromosomal damage even at very high radiation doses.     

Effect of Irradiation of the Primer DNA on the Incorporation of Precursors

The rate of enzymatic incorporation of ³H-labelled thymidine into primer DNA was studied after the DNA had been irradiated with doses up to 360 000 roentgens of gamma-rays. Non-irradiated DNA-synthesizing enzyme obtained from regenerating rat-liver was used together with DNA prepared by the method of Kay, Simmons and Dounce (1952) in the incubation system described by Bollum (1959). An initial increase followed by a decrease was noted in one sample of DNA, whereas a second less-highly-polymerized sample showed only a decrease in the rate of incorporation. Isolated calf-thymus nuclei showed no changes in the rate of incorporation of ³H-labelled thymidine or cytidylic acid after irradiation with very high doses of gamma-rays. The results of these in vitro studies do not provide support for the concept that damage to DNA preventing replication is a factor in the decrease in DNA-synthesis noted in irradiated cells.     

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.     

Three-dimensional dose-distribution measurement of therapeutic carbon-ion beams using a ZnS scintillator sheet

The accurate measurement of the 3D dose distribution of carbon-ion beams is essential for safe carbon-ion therapy. Although ionization chambers scanned in a water tank or air are conventionally used for this purpose, these measurement methods are time-consuming. We thus developed a rapid 3D dose-measurement tool that employs a silver-activated zinc sulfide (ZnS) scintillator with lower linear energy transfer (LET) dependence than gadolinium-based (Gd) scintillators; this tool enables the measurement of carbon-ion beams with small corrections. A ZnS scintillator sheet was placed vertical to the beam axis and installed in a shaded box. Scintillation images produced by incident carbon-ions were reflected with a mirror and captured with a charge-coupled device (CCD) camera. A 290 MeV/nucleon mono-energetic beam and spread-out Bragg peak (SOBP) carbon-ion passive beams were delivered at the Gunma University Heavy Ion Medical Center. A water tank was installed above the scintillator with the water level remotely adjusted to the measurement depth. Images were recorded at various water depths and stacked in the depth direction to create 3D scintillation images. Depth and lateral profiles were analyzed from the images. The ZnS-scintillator-measured depth profile agreed with the depth dose measured using an ionization chamber, outperforming the conventional Gd-based scintillator. Measurements were realized with smaller corrections for a carbon-ion beam with a higher LET than a proton. Lateral profiles at the entrance and the Bragg peak depths could be measured with this tool. The proposed method would make it possible to rapidly perform 3D dose-distribution measurements of carbon-ion beams with smaller quenching corrections.     

Allelic imbalance at 11p15.5-15.4 correlated with c-Ha-ras mutation during radiation-induced neoplastic transformation of human breast epithelial cells

Breast cancer is the most frequent malignancy in women throughout much of the developed world and is associated with a multistage process involving a number of genetic mutations and their corresponding cellular phenotypic alterations. It has already been shown that neoplastic transformation of a spontaneously immortalized human breast epithelial (MCF-10F) cell line by radiation, in combination with estrogen, represents a successful model in studying the molecular and biological alterations that may contribute to the tumorigenic process. In the present study, the incidence of allelic alterations (microsatellite instability/loss of heterozygosity) on chromosome 11 in different radiation-induced primary and secondary tumorigenic cell lines, relative to the control MCF-10F cells was investigated. We identified 3 regions of the chromosome 11 (11p15-p15.5, 11q13 and 11q23) that showed high incidence of LOH among these tumor cell lines and suggested a potential role for these chromosomal regions in breast carcinogenesis. Among them, locus 11p15.5, where c-Ha-ras oncogene is located, had incidence of allelic imbalance between 25-40%. Furthermore, direct sequencing analysis of codons 12 and 61 of the c-Ha-ras oncogene identified various point mutations. These data highlight the importance of chromosome 11 in radiation induced malignant transformation of human breast epithelial cells and suggest the usefulness of the model in uncovering specific derangements during breast cancer progression.     

Biological effectiveness of 12C and 20Ne ions with very high LET

Purpose: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of ¹²C and ²⁰Ne ions.

Materials and methods: Chinese hamster ovary (CHO-K1) cells were exposed to high LET ¹²C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and ²⁰Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up.

Results: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440–830 keV/μm for ¹²C and for 1020–1600 keV/μm for ²⁰Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET.

Conclusions: The RBE decreases with increasing LET in the range between 440 and 1600 keV/μm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.     

A small fish model for quantitative analysis of radiation effects using visualized thymus responses in GFP transgenic medaka

Abstract

Purpose: The aim of this study was to establish a new method of real-time, in vivo detection of radiation damage and recovery.

Methods: The thymus was observed under fluorescent light in a green fluorescent protein transgenic medaka. After irradiation, medaka thymus images were analyzed to quantify the effects of radiation by measuring changes in thymus size. A single acute irradiation of X-rays (0–30?Gy) or heavy Fe ions (0–10?Gy) was delivered to the medaka. Images were captured 0, 1, 2, 3, 5, 7, 11, and 21 d after irradiation. Dose-response assessment was conducted to provide a direct measurement of the effects of the radiation.

Conclusion: A biomonitoring system to detect the effects of radiation in real time was established. Using this system, the threshold doses for the induction of thymic atrophy by acute X-rays and Fe ions were 2–5?Gy and 0.5–1?Gy, respectively. The Relative Biological Effectiveness (RBE) of Fe-ion to X-rays was estimated to be around 3. This system may be used to evaluate the risk from concurrent exposure to hazards, such as chemicals and radiation, and for aging research.     

Malignant salivary gland tumours: Can fast neutron therapy results point the way to carbon ion therapy?

Background and purpose

To evaluate the outcome of malignant salivary gland tumours treated with neutron therapy to assess the potential for other high linear energy transfer (LET) beams.

Materials and methods

Neutrons at iThemba LABS are produced by the reaction of 66 MeV protons on a beryllium target. A median dose 20.4 Gy, in 12 fractions in 4 weeks or 15 fractions in 5 weeks, was given to 335 patients with 176 irresectable, 104 macroscopically residual and 55 unresected tumours.

Results

Locoregional control was 60.6% at 5 years and 39.1% at 10 years and DSS was 66.8% and 53.7% at 5 and 10 years respectively.In the univariate analysis T4, >4 cm, high grade, squamous carcinoma, unresected and irresectable tumours, and positive nodes were significantly worse for LRC. In the multivariate analysis tumours >6 cm, squamous carcinoma, irresectable tumours and nodes were significantly worse for LRC. Tumours >6 cm, high grade, squamous carcinoma and nodes were significantly worse for DSS. Neither LRC nor DSS was influenced by age, sex, site, dose, fractionation or for initial or recurrent disease.

Conclusions

Neutron therapy appears to be the treatment of choice for macroscopically incompletely excised and irresectable salivary gland tumours with improved survival rates. Further improvement may be achieved with other high LET modalities with a superior dose profile, such as carbon ions.