Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability

Abstract

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

Fig 1. Representative histogram plots show CD3 expression levels measured by FACS.

The percentage shift in CD3-FITC fluorescence (overlaid as a black line) was calculated relative to isotype controls (coloured grey). The histogram peaks are typically broad due to the population being of mixed cell type. CD3 expression levels before T-cell depletion of BMNC (Tw) show a mature T-lymphocyte population of 21% (A). Complete depletion of CD3⁺ cells was observed in T-cell depleted (Td) fractions on day 0 (B). A new T-cell subpopulation, accounting for 54% of Td sample, was produced by day 12 (C). Td T-cell proportion was increased over 10 days of long-term culture to reach 68% on day 25 (D). No CD3⁺ cells were observed in Td, Tp or Tw populations cultured in the absence of stimulatory factors PHA and IL-2. Td data only shown in (E).

Fig 2. Representative dot-plots showing co-expression measured by FACS.

Mononuclear cell sub-populations were gated and representative dot-plots produced showing levels of CD3 and CD4 co-expression measured by FACS. Representative histogram data for the same time-points show CD4 and CD8 expression levels in un-gated populations. The percentage shift in CD4-PE (coloured grey) or CD8-PE fluorescence (overlaid as a black line) was calculated relative to isotype controls (not shown). On day 12, 4% of mature CD3⁺ T-lymphocytes in Tw populations co-express the CD4 cell surface marker (A) compared with 91% in Td populations (B). CD8⁺ cells predominate in Tw samples (C). CD4⁺ cells predominate in Td samples (D).

Fig 3. M-FISH karyotype of clonal complex exchange.

M-FISH karyotype (A) and cartoon (B) showing the clonal complex exchange t(2;4;17) that was observed Td populations previously exposed to 0.5 Gy α-particles. The minimum number of chromosome breaks required to form the observed exchange are shown. Representative M-FISH karyotypes of the clonal complex that also contain additional non-clonal abnormalities such as chromosome breaks (del(2q) (B)) and simple translocations (t(6;18) (C)).