RBE of heavy ions for producing chromosome aberrations in human lymphocytes: metaphase versus G2-PCC analysis

The analysis of chromosome aberration in peripheral blood lymphocytes is a sensitive and frequently applied method to assess the individual dose following accidental, occupational or medical exposure to ionizing radiation. According to the standard protocol [1], lymphocytes are cultured in vitro for 48 h. Then, metaphases are harvested and chromosome aberrations are scored with the expectation that the data are representative of the whole cell population. However, we and others have shown that this protocol is not reliable in the case of high LET exposure [2, 3 and references therein]. Heavy ions induce a severe G2-arrest and the measurement of aberrations in metaphases at 48 h will result in very low RBE estimates, because heavily damaged cells are drastically delayed in cell cycle and are not included in the analysis. To overcome this problem, the measurement of aberrations in G2-cells collected at 48 h by chemically-induced premature chromosome condensation (PCC) has been proposed [4]. However, up to now little is known about the LET-dependence of the G2-arrest in human lymphocytes and it has not been experimentally demonstrated to what extent the PCC-assay at 48 h post-irradiation accounts for high LET induced cell cycle delay of heavily damaged lymphocytes. To clarify this point we exposed lymphocytes of a healthy volunteer to heavy ions (C-, Fe-, Xeand Cr-ions) or X-rays. Aberration yields were measured at several sampling times (48, 60, 72 and 84 h) in both first cycle G2-PCC and metaphase cells and RBE values for the induction of 1 aberration per cell were derived (for details see [3]). As shown in Fig. 1 (a), RBE estimates derived from metaphase data at 48 h increased with LET, reached a maximum around 155 keV/μm and decreased with a further rise in LET. In addition, for cells exposed to particles with LET>30 keV/μm, higher RBE values were derived at later sampling times due to the mitotic delay of heavily damaged cells. This effect was most pronounced after exposure to 175 keV/μm C-ions. Accordingly, Fig. 1 (b) shows the RBE values obtained for G2-PCC cells. As observed for metaphase cells, the RBE peaked at 155 keV/μm. However, in contrast to metaphase analysis the RBE estimates from PCC-samples declined less steeply with increasing LET. Furthermore, the time-dependent changes of the RBE values were smaller for G2-PCC analysis than for metaphase analysis. Altogether our data demonstrate that the standard analysis of aberrations in metaphase cells 48 h postirradiation will considerably underestimate the RBE of high LET radiation. Scoring of aberrations in G2-PCCs at 48 h accounts for most, but not all, of high LET induced delay of severely damaged cells. However, when particles with LET>150 keV/μm are applied, still a fraction of multiple damaged cells escape detection by G2-analysis 48 h post-irradiation. Thus, in case of a suspected exposure to particles with a very high LET aberrations should be measured at a second (later) sampling time even if the G2PCC assay is applied. For a more detailed discussion see [3].