Comparative RNA-Seq transcriptome analyses reveal dynamic time dependent effects of 56Fe, 16O, and 28Si irradiation on the induction of murine hepatocellular carcinoma

One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z>13) which can lead to induction of hepatocellular carcinoma (HCC). We performed comparative RNA-Seq transcriptomic analysis to assess the carcinogenic effects of 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) ions in a mouse model for radiation-induced hepatocellular carcinoma. C3H/HeNCrl mice were subjected to total body irradiation to simulate space HZE-irradiation environment and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent gradual carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the effects of these HZE ions, particularly immunological, on the carcinogenic process of HCC, suggesting different molecular mechanisms of tumorigenesis. Also seen in this study was novel unmapped transcripts that were significantly affected by HZE. To investigate the biological functions of these transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes. Taken together, these findings not only led to a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation but also have important implications for discovery of potential countermeasures and identification of biomarkers of HZE-induced HCC. Overall design: C3H/HeNCrl mice purchased from Charles River (Wilmington, MA) were used in this experiment. Mice were used for this study because they have been shown in the past to be a good experimental model for liver carcinogenesis. Strains used were based on previous studies by Weil et al. (2009) demonstrating that C3H/HeNCrl mice are sensitive to the induction of HCC after exposure to a dose of 0.2 Gy of 600 MeV/n 56Fe. Tumor induction studies and studies of molecular changes in the irradiated tissues can only be conducted in whole animals. Conducted studies were approved by the institutional animal care and use committees (IACUCs) that are charged with evaluating the appropriateness of the use of animals in specific experiments and the numbers of animals requested for each group. The numbers of animals used were based on the expected numbers of irradiation-related tumors that would develop if animals were allowed to live out their lifespan. Power calculations for numbers in this study are based on chi-square test for comparing two proportions, controlling two-sided significance level at 0.05 or with 95% confidence about our results, and with 80% power. Computer models or cell culture studies are not possible based on an extensive literature search. Serial sacrifice study consisted of 15 male mice with 3 mice per time point and five time-points consisting of 30, 60, 120, 270, and 360 days post-exposure to HZE: 56Fe, 16O, and 28Si irradiation. Additionally, 15 mice were used as controls resulting in a total of 60 eight to ten-week-old male mice for this study. The 3 treatment groups were: 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), 350 MeV/n 28Si (0.2 Gy), and no irradiation. The mice were shipped from the vendor to Brookhaven National Laboratories (BNL) and housed at the BNL animal facility until the time of irradiation at the NASA Space Radiation Laboratory (NSRL). Following irradiation, the animals were shipped to the UTMB Animal Resources Center (ARC), quarantined for one month and then maintained in the ARC for the duration of the experiment. Animals were housed in sterile cages with free access to food and water. Facilities at both BNL and UTMB are fully AAALAC accredited which ensured adequacy of all animal care issues at both animal facilities. At each of the five time-points, 3 animals from each treatment group were randomly selected and euthanized by using CO2 asphyxiation as per current AVMA guidelines for euthanasia. Prior to euthanasia animals were weighed and weight recorded. Post euthanasia tissues of the left lobe of livers were collected, snap-frozen on either dry ice or liquid nitrogen and stored at -80°C until tissues could be extracted for RNA. Livers were sampled by taking two 40-micron thick slices using a cryotome at -20°C.