Global transcriptomic analysis suggests carbon dioxide as an environmental stressor in spaceflight: A systems biology GeneLab case study

www.nature.com/scientificreports OPEN Received: 30 November 2017 Accepted: 26 February 2018 Published: xx xx xxxx Global transcriptomic analysis suggests carbon dioxide as an environmental stressor in spaceflight: A systems biology GeneLab case study Afshin Beheshti1, Egle Cekanaviciute 2 , David J. Smith3 & Sylvain V. Costes 3 Spaceflight introduces a combination of environmental stressors, including microgravity, ionizing radiation, changes in diet and altered atmospheric gas composition. In order to understand the impact of each environmental component on astronauts it is important to investigate potential influences in isolation. Rodent spaceflight experiments involve both standard vivarium cages and animal enclosure modules (AEMs), which are cages used to house rodents in spaceflight. Ground control AEMs are engineered to match the spaceflight environment. There are limited studies examining the biological response invariably due to the configuration of AEM and vivarium housing. To investigate the innate global transcriptomic patterns of rodents housed in spaceflight-matched AEM compared to standard vivarium cages we utilized publicly available data from the NASA GeneLab repository. Using a systems biology approach, we observed that AEM housing was associated with significant transcriptomic differences, including reduced metabolism, altered immune responses, and activation of possible tumorigenic pathways. Although we did not perform any functional studies, our findings revealed a mild hypoxic phenotype in AEM, possibly due to atmospheric carbon dioxide that was increased to match conditions in spaceflight. Our investigation illustrates the process of generating new hypotheses and informing future experimental research by repurposing multiple space-flown datasets. Comprehensive analysis of molecular signatures, such as transcriptional profiling, has become a standard technique in space biosciences and typically generates more extensive data than is required for the specific topic of investigation. Making all spaceflight data publicly accessible ensures that biological experiments can be repurposed to answer novel research questions and generate hypotheses. Therefore, the GeneLab open science platform (genelab.nasa.gov) was created to store raw molecular “omics” data from ground and spaceflight biology experiments supported by NASA. Here we present a case study using GeneLab datasets generated from ground controls associated with multiple rodent spaceflight datasets. Our overarching aim is to generate a hypothesis to drive future spaceflight rodent research and examine the potential impact of one known confounding factor in spaceflight, i.e. the environment in the animal habitat. We proceeded by incorporating multiple, independent publicly available transcriptomic datasets from spaceflight experiments. Investigating spaceflight-induced changes in the transcriptome involves sending model organisms to orbit, such as rodents on the space shuttle (Space Transportation System program, STS), on satellites such as Bion-M1 (BF), or on the International Space Station (ISS). In these experiments multiple aspects of the environment are collectively altered. NASA space flown rodents are housed in a specific type of cage, called the Animal Enclosure Module (AEM). Within an AEM, animals will experience gravitational changes ranging from 1 Wyle Labs, Space Biosciences Division, NASA Ames Research Center, Mountain View, CA, USA. 2Universities Space Research Association, Space Biosciences Division, NASA Ames Research Center, Mountain View, CA, USA. 3NASA, Space Biosciences Division, NASA Ames Research Center, Mountain View, CA, USA. Afshin Beheshti and Egle Cekanaviciute contributed equally to this work. Correspondence and requests for materials should be addressed to A.B. (email: ) or S.V.C. (email: ) SCiENtifiC REportS | (2018) 8:4191 | DOI:10.1038/s41598-018-22613-1 1 www.nature.com/scientificreports/ Figure 1. Illustration of the AEM and vivarium cages and GeneLab datasets used for analysis. (A) Images and dimensions of the both the AEM and vivarium cages used to house rodents. The upper two photos of the AEM cage was provided by NASA (Credits: NASA/Dominic Hart and https://www.nasa.gov/ames/research/ space-biosciences/rodent-research-1). The vivarium cage photo was taken in our laboratory. (B) The GeneLab datasets which were used for analysis including the information on rodent species, the specific tissue type and its approximate location in a rodent, duration of experiment, and CO2 levels in the AEM cage. hypergravity during launch and landing to microgravity in orbit while simultaneously exposed to higher levels of ionizing radiation than found on Earth1. Selecting appropriate controls for such multifactorial experiments is therefore complicated. The most frequently used experimental design is to keep all environmental conditions the same except for the flight by containing rodents in an AEM on spacecraft and using the same type of AEM hardware for ground controls2. An alternative and complementary approach is using regular vivarium cages for housing rodents as controls. AEM has been used as the standard rodent enclosure without major modifications in spaceflight experiments from STS2 to Bion-M1 (BF)3, and its more modern version called the Habitat module of the Rodent Research Hardware System is currently used on the ISS. Both AEM and vivarium housing follow the standard guidelines for laboratory animal care, which require at least 15 square inches per >25 g adult rodent (NASA Johnson Space Center Animal Care and Use Handbook). AEM can either contain up to 10 mice in two compartments (5 mice per compartment) or 6 rats maximum (3 rats per compartment). This is in comparison to the vivarium cages which can house either 5 mice, or 2 rats maximum in a single compartment (depending on the rat’s mass). The AEM has a combined larger surface area per rodent, because it includes climbable walls throughout the enclosure (Fig. 1A). Rodents housed in AEM and vivarium cages are typically kept on the same light/dark cycle and the air has the same oxygen concentration. However, CO2 concentration in AEM ground controls replicates CO2 concentration on spacecraft2,4, which tends to be up to an order of magnitude higher than on Earth. In general, CO2 concentration on ground and therefore, in vivarium cages is approximately 300 ppm, while on the space shuttle and in matched ground AEM it reaches up to 3000 ppm5. Notably, it was lower in the AEM controls for the Bion-M1 satellite study: 682 ppm on average with 718 ppm standard deviation (range: 201–2096 ppm)4. Interestingly, previous studies on different CO2 conditions on human health have revealed major impact on cognitive functions. Specifically, it was observed that a 400 ppm increase in CO2 levels results in a 21% drop in cognitive scores6 and specific research related to the increased CO2 levels on the ISS have shown increa (...truncated)