Munira A. Kadhim1,2, Lora M. Green1, Daila S.
Gridley1, Deborah K. Murray1, DaThao Tran1,
Debbie Bowler2, Denise Macdonald2, MelbaAndres1,
Michael F. Moyers1 Dudley T. Goodhead2 & Gregory
A. Nelson1
1Radiobiology Program-Radiation Medicine Loma Linda University
Chan Shun Pavilion room A1010, 11175 Campus Street Loma Linda, CA 92354.
2MRC Radiation and Genome Stability Unit Harwell, Didcot,
Oxon OX11 ORD United Kingdom
m.kadhim@har.mrc.ac.uk
Introduction
The assembly of the International Space Station (ISS) is expected to require 1000 hours of extravehicular activity (EVA) during a period of high solar activity. Exposure to galactic cosmic rays (GCR) and solar particle events (SPE) are a major health risk to humans. Protons and high Z, energetic particles (HZE) comprise the GCR spectrum and may exert considerable biological effects even at low fluence. During the construction of the ISS the astronauts could be exposed to substantial levels of protons and electrons from SPE and electron belt enhancement [Space Rad Health Vol1 (1) 2001]. To estimate the protective threshold limits provided by the EVA suits, the US and Russian extravehicular maneuvering units (EMU) were subjected to protons and electrons at Loma Linda University Medical Center (LLUMC) facilities in California.
We conducted in vitro biological experiments using murine hematopoietic stem cells to investigate whether the helmet of the US EMU would provide protection from the damaging effects of radiation relevant to those encountered in space. Recent studies have shown that in addition to acute effects of radiation that occur within a few cell divisions, longer-term changes occur in the surviving cells leading to pronounced chromosomal instability [Kadhim et al. 1992, Nature; Kadhim et al. 1995 Int J Radiat Biol; Kadhim et al. 2001Rad. Res.]. However, and perhaps more importantly, there are genetic components in mouse and man that determine the relative sensitivity of their cells to radiation-induced genomic instability and ultimately their cancer risk. Additionally, there are untargeted genetic lesions, where the induced “mutation rate” at specific loci is higher than can be attributed to direct radiation-induced DNA damage, which may in part be due to transmitted genetic instability via a bystander effect mechanism. Despite many high profile studies, little is known about either the underlying mechanisms or the in vivo consequences of such induced phenotype(s).
Methods
Bone marrow cells isolated from two inbred strains of mice, known to be sensitive (CBA) or resistant (C57BL/6) to high LET radiation induced delayed expression of chromosomal/genomic instability [Watson et al. 1997 Int J Radiat Biol] were exposed to proton radiation at doses of 0, 0.5, 1 and 2 Gy while shielded or unshielded by the US helmet. Cultures were established and samples taken at early (1-2 cell divisions) and late (12-15 divisions) times post irradiation. Analysis included: cytogenetic aberrations [methods previously described in: Kadhim et al. 1992, Nature], apoptosis by Annexin V binding and BrdU incorporation [methods previously described in: Green et al. 2001 Radiat Res], and TNF-a, IL-1b, IL-2, GM-CSF and TGF-b1 levels released to the culture supernatant [methods previously described in Gridley et al. 1996 Int J Oncol].
Results
Cytogenetic Analysis - Bone marrow stem cells from both CBA and C57/B mice had significant induction of chromosomal aberration when assessed at early time post irradiation. These aberrations were detected in all cultures and at all doses whilst either inside or outside the EMU helmet. There was a reduction in the frequency of aberrations at early time points in culture from both strains, when irradiated outside the helmet that increased with increasing dose of radiation. Conversely, there was no dose relationship observed for the induction of chromosomal aberration for those early samples that irradiated inside the helmet. After 12-15 population doublings, the proportion of chromosomally aberrant cells from both strains of mice, were highly significant compared to the non-irradiated control group. This finding was consistent for low doses and under both irradiation conditions. The expression of instability however, declined in the 2Gy proton-irradiated groups under both conditions, but the levels were still significantly higher than control value.
The major cytogenetic aberrations (chromatid breaks chromosome fragments) were consistent with the transmission of chromosomal instability. Moreover, the frequency of induced instability phenotype was considerably greater than the frequency of induced mutations at specific loci which is characterized by a higher ratio of chromatid to chromosome-type aberrations (Kadhim et al. 1992).
Apoptosis- The sum of annexin V binding (early apoptosis) and BrdU incorporation (late apoptosis) were used to reflect the total apoptosis in the bone marrow cultures. At early times post irradiation the highest apoptosis occurred in both strains at the lower doses and was higher in those cultures exposed while inside the EMU helmet. The apoptosis measured in the cultures harvested at the late time point were again higher at the lower doses but the cultures exposed outside the EMU helmet were slightly more elevated than those shielded by the helmet. C57 mouse marrow had a near 40% higher level of apoptosis at 1 Gy than the CBA mouse marrow cultures. There was no linearity to the dose response and the peak level of apoptosis was not equal for the two strains. The level of apoptosis at 2Gy was declining for all conditions (strains, doses, times and shielded/ unshielded).
Cytokine Production/Release in Irradiated & Control Culture Supernatants- TGF-b1 concentrations increased dramatically in supernatants of cells from both strains of mice compared to non-irradiated cultures under certain conditions. With CBA/Ca cells, the greatest increase in TGF-b1 was seen at 7 days after a shielded dose of 0.5 Gy (23-fold higher than 0 Gy), whereas the peak level with C57BL/6 cells occurred at 48 hr following an unshielded dose of 1 Gy (16.5-fold higher than 0 Gy). Changes in TNF-a expression were also observed, but to a much lesser degree than with TGF-b1. With CBA/Ca cells, the greatest increase was found at 7 days following a dose of 0.5 Gy (2.0-fold higher than 0 Gy); the highest levels in supernatants from C57BL/6 cells occurred at 7 days after a 2 Gy shielded dose of radiation (3.6-fold higher than 0 Gy). IL-1, IL-2 and GM-CSF were below detectable levels in supernatants from non-irradiated controls, as well as those from irradiated cells, regardless of shielding.
Conclusions