Liver enzyme activity assays

Protein was extracted from an aliquot of each liver sample from Basal, GC, and FLT Experimental mice (n = 5 from each group), and from Basal, Viv, GC, and FLT Validation mice (n = 2 for the tissues that were dissected either on the ground or on-orbit then frozen; and n = 5 (out of 8) for the samples that were dissected from frozen carcasses from each group), as well as their respective positive controls (n = 5 for Experimental control and n = 3 for Validation control). Activities of three enzymes were determined using the method previously described⁵⁷. Briefly, catalase activity was measured using the colorimetric assay according to the manufacturer’s instructions (Oxford Biomedical Research, Oxford, MI), and normalized to total protein levels (Thermo Scientific™ Pierce™ Micro BCA™ Protein Assay Kit, Pierce Biotechnology, Rockford, IL). Catalase protein levels were measured using the Catalase Human ELISA Kit (Abcam, Cambridge, MA), and specific activity of the enzyme was calculated (U/μg catalase protein). GSR activity levels were determined using the GSR Assay Kit (Abcam, Cambridge, MA) and GAPDH activity levels were determined using the colorimetric GAPDH assay kit (ScienCell Research Laboratories, Carlsbad, CA).