Countering Low-Linear Energy Transfer (LET) and High-LET Radiation Exposure with Pro-Methyl Hoechst (PMH) and EUK-134 (Mg-Salen Complex): Radioprotection of Normal Human CD34+ Cells in a Simulated Deep Space Environment.
Authors: Gangenahalli Gurudutta U. , Millholland John M. , Kalota Anna , Bennett Paula , Sutherland Betsy M. , Gewirtz Alan M. 
Published: April 2020

Abstract
The deep space radiation environment is composed of both low-, and high-, LET radiation sources. High-LET radiation particles (HZE) are particularly dangerous because, by direction ionization of DNA, or radiolysis of water, they induce free radical formation that causes relatively non-repairable double strand breaks (DSB) and other complex damages to cellular DNA. The risk to flight crews exposed to HZE on extended duration voyages, such as the proposed Mars mission, has not been quantitated. Crew safety, and mission planning, dictate that such studies be done. We are examining the effects of low and high-LET radiation on human hematopoietic stem and progenitor cell function using normal human CD34+ cells obtained from consenting donors. Low-LET radiation was derived from a 137Cs source while HZE, primarily in the form of radioactive Fe, Ti, Si were generated by the Brookhaven National Laboratory’s (BNL) relativistic heavy ion collider-alternating gradient synchrotron (RHIC-AGS). Cells were prepared at UPENN, and shipped to BNL by overnight courier. The cells were then irradiated, with or without candidate radioprotectants, and aliquots of cells were then analyszed for DNA damage, and colony forming unit (CFU) function. Our initial studies indicated that human CD34+ cells demonstrated a dose dependent sensitivity to exposures as low as 15 cGy of both radiation types. The effects of HZE particles were more severe as shown by the dramatic decrease in assayable BFU-E, CFU-E, CFU-GM and CFU-GEMM assays. PMH treatment prior to irradiation, enhanced CFU formation by all lineages from 2–4 fold in the dose range of 15–50 cGy (137Cs) as compared to untreated cells. When doses increased to 70 cGy, PMH still displayed significant radioprotective effects in BFU-E and CFU-GEMM assay, as much as 2–3 fold, indicating some lineage specificity to its protective abilities. EUK-134 displayed significant radio-protection between 15-30 cGy as well and was slightly additive when combined with PMH. CFU were increased several fold compared to untreated controls in the dose range of 30-50 cGy of Fe. At 50 cGy, CFU-E, and CFU-GM were no longer protected. A decrease in the levels of phosphorylated histone H2A.X, a sensor of DSB, was observed in cells treated with PMH, and EUK-134, and correlated with the radioprotection observed. Immunochemical staining also documented an increase in repair proteins unique for DSB, such as Ku70/80, MRE-11 and Rad-50. Finally, the number of DNA ends labeled with dUTP-(FITC) terminal transferease enzyme was also increased. Altogether, these observations suggest that between 15–50 cGy, PMH and EUK-134 prevent DNA damage and DSB formation, thereby preserving CFU compared to untreated controls. Protective effects are lost when doses were greater than 50 cGy. We conclude that PMH and EUK-134 is effective against both low and high-LET, and that similar radioprotective mechanisms may be triggered in cells by each. Experiments to address the precise mechanisms of radioprotection by these compounds are ongoing in our labs, and will hopefully assist in the development of even more efficient radioprotective agents