The time-course of Fe-ion (200 MeV/u, 440 keV/ μm) and X-ray induced chromosomal damage was investigated in human lymphocytes. After cells were expos

Purpose: To examine the relationship between cell proliferation and the expression of chromosomal damage in normal human skin fibroblasts after X‐ray and particle irradiation. Materials and methods: Confluent G0/G1 AG1522B cells were exposed to X‐rays or 195 MeV u⁻¹ C ions with a linear energy transfer of 16.6 keV µm⁻¹ in the dose range 1–4 Gy. Directly after irradiation, cells were reseeded at a low density in medium containing 5‐bromo‐2′‐deoxyuridine. At multiple time points post‐irradiation, the cumulative BrdU‐labelling index, mitotic index and aberration frequency were measured. Based on these data, the total amount of damage induced within the entire cell population was estimated by means of mathematical analysis. Results: Both types of radiation exposure exert a pronounced effect on the cell cycle progression of fibroblasts. They result in delayed entry of cells into S‐phase and into the first mitosis, and cause a dramatic reduction in mitotic activity. Measurement of chromosomal damage in first‐cycle cells at multiple time points post‐irradiation shows that the frequencies of aberrant cells and aberrations increase with time up to twofold for the lower doses. However, for the higher doses, this effect is less pronounced or even disappears. When the data for the whole cell population are analysed, it becomes evident that only a few damaged fibroblasts can progress to the first mitosis, a response attributable at least in part to a long‐term arrest of injured cells in the initial G0/G1‐phase. As observed in other investigations, the effectiveness of 195 MeV u⁻¹ C ions was similar or slightly higher than X‐rays for all endpoints studied leading to a relative biological effectiveness in the range 1.0–1.4. Conclusions: Cell cycle arrests affect the aberration yield observable in normal human fibroblasts at mitosis. The data obtained for the cell population as a whole reveal that injured cells are rapidly removed from the mitotically active population through a chronic cell cycle arrest, which is consistent with other studies that indicate that this response is a specific strategy of fibroblasts to minimize the fixation and propagation of genetic alterations.

Purpose : To investigate further the effect of linear energy transfer (LET) on the yield and quality of aberrations at different post-irradiation sampling times. Materials and methods : V79 G 1 -cells were exposed to either 10.6 MeV u -1 Ne ions (360 keV μ m -1) or 11.1 MeV u -1 Kr ions (3980 keV μ m -1) and chromosomal damage was measured in metaphase cells at several 2-h sampling intervals up to 30 h post-irradiation. To differentiate between cells in the first and second post-irradiation cycle, the fluorescence-plus-Giemsa technique was applied. Results : In both experiments, an increase in the yield of aberrant cells as well as the number of aberrations per cell was observed in first- and second-cycle metaphases. Yet, the increase in the number of aberrations per cells was more pronounced for Kr ions and at comparable fluences Kr ions produced more aberrations than Ne ions. Because no sampling time was representative for the whole cell polpulation, the total amount of Ne and Kr ion-induced chromosomal damage was determined by means of a mathematical approach and used for the comparison of data. Furthermore, in accordance with previous studies, LET-dependent changes in the spectrum of aberration types were detected, i.e. with increasing LET a higher fraction of chromatid-type aberrations was observed, although cells had been exposed in G 1. In addition, more chromosomal breaks and less exchange-type aberrations were found. Conclusions : The observation that cell-cycle progression is related to the amount of aberrations harboured by a cell demonstrates that the routinely applied method to measure aberration frequencies in metaphase cells at only one post-irradiation sampling time will unavoidably result in an under- or overestimation of the cytogenetic effects of particles. Consequently, for a meaningful quantification of chromosomal damage, multiple fixation regimes should be used so that the complete time-course of aberrations can be taken into account. Moreover, to avoid bias, all aberration types should be recorded and included in the analysis since the aberration spectrum changes with LET.

Microelectrode array (MEA) technology in combination with three-dimensional (3D) neuronal cell models derived from human embryonic stem cells (hESC) provide an excellent tool for neurotoxicity screening. Yet, there are significant challenges in terms of data processing and analysis, since neuronal signals have very small amplitudes and the 3D structure enhances the level of background noise. Thus, neuronal signal analysis requires the application of highly sophisticated algorithms. In this study, we present a new approach optimized for the detection of spikes recorded from 3D neurospheres (NS) with a very low signal-to-noise ratio. This was achieved by extending simple threshold-based spike detection utilizing a highly sensitive algorithm named SWTTEO. This analysis procedure was applied to data obtained from hESC-derived NS grown on MEA chips. Specifically, we examined changes in the activity pattern occurring within the first ten days of electrical activity. We further analyzed the response of NS to the GABA receptor antagonist bicuculline. With this new algorithm method we obtained more reliable results compared to the simple threshold-based spike detection.

Purpose : As an extension of previous studies, the time-course of high-LET-induced chromosomal damage was investigated in first- and second-cycle V79 Chinese hamster cells. Materials and methods : Cells were exposed in G 1 to 10.4MeV/u Ar ions (LET=1226keV/mum) and chromosomal damage was measured at 2h sampling intervals between 10h and 34h after irradiation. To distinguish between cells in different post-irradiation cycles, the fluorescence-plus-Giemsa technique was applied. Results : For first- and second-generation cells, the number of aberrant metaphases and aberrations per metaphase were found to increase markedly with sampling time, demonstrating that cell cycle progression was delayed according to the number of lesions carried by the cell. To account for the time-dependent expression of chromosomal damage a mathematical approach was used based on the integrated flux of aberrant cells entering mitosis. Moreover, the analysis of Ar ion-induced chromosome lesions confirmed that high-LET radiation results in specific changes in the spectrum of aberration types. In particular, an increased rate of chromatid-type aberrations as well as a high frequency of chromosomal breaks was found, although the cells were exposed in G 1. Conclusions : Due to the fact that cells collected at one sampling time are not representative of the entire population, the complete time-course of chromosomal damage has to be taken into account for the determination of a meaningful RBE value. Otherwise, the analysis of chromosomal damage can result in a pronounced over- or underestimation of the RBE depending on the subpopulation of cells entering mitosis at that particular sampling time.