This study quantitatively characterized environmental parameters in the DUGL at the CJEM and investigated the biological effects of these environmental parameters on V79 cells. This study provides the first research data to inform the new discipline of deep-underground medicine.

Six environmental parameters (radon gas, O2, total γ ray dose rate, CO2, air pressure, relative humidity) with potential biological effects were monitored in the DUGL and the AGL. Relative humidity (99%), air pressure, and concentration of CO2 and radon gas were significantly higher in the DUGL compared to the AGL. The total γ radiation dose rate was significantly lower in the DUGL (0.03–0.05 μSv/h) compared to the AGL (0.13–0.18 μSv/h), even though radon gas is an important source of ionizing radiation. Compared to the LNGS, the concentration of radon gas was slightly higher in the DUGL at the CJEM, but total γ radiation dose rate and relative humidity were similar. Radon concentration in the DUGL was 1.5 pCi/L, which is less than normal background (1.7 nGy/h)10.

The present study confirmed previous reports that show reduced growth rates in cell lines within a short time (several days to two weeks) of being introduced to the deep underground6,7,8,15. Findings contrast with Satta et al. who found a significant increase in cell density at confluence in V79 cells grown in the LNGS compared to parallel populations cultured above ground9,16. These disparate results may be explained by dissimilar methodology. In the present study, cell proliferation was measured daily during the 7 days after V79 cells had been introduced to the DUGL, while Satta et al. observed their cells when they had been maintained in exponential growth in the LNGS for 9 months9,16. Short term stress responses in cells undergoing an acute environmental change differ from the adaptive response seen in cells exposed to chronic stress17. Cells cultured in the deep underground for many months may adapt to their environment and show no difference in proliferation rates compared to cells grown above ground9,16.

We speculate that reduced background radiation inhibited V79 cell proliferation in the DUGL at the CJEM. The rock cover over the DUGL provides shielding equivalent to 4,000 m of water, which almost completely eliminates cosmic radiation18. Terrestrial radiation is emitted from natural radio nuclides present in varying amounts in the soil, air, water and other environmental materials. Radon, including 222Rn and 220Rn derived from terrestrial radioactive elements of uranium and thorium, is the most important component of natural radiation. Radon gas concentration was significantly higher but the γ radiation dose rate was significantly lower in the DUGL compared to the AGL. Other environmental parameters, including light, O2 levels, relative humidity, temperature, concentration of CO2 and air pressure can affect cell proliferation, but were unlikely to influence cell growth in the DUGL. Light, O2 levels, humidity, temperature, and concentration of CO2 were maintained at the same levels inside the CO2 incubators used for cell culture in the DUGL and the AGL. Air pressure could have affected biomass yield in cell cultures as cell growth rate is enhanced at 1.2–6 bar19,20. Air pressure in the DUGL was slightly higher than the AGL; however, the difference was reduced by the shift of gas and liquid as the cells were cultured in liquid. As much as possible to decrease batch effects, the analyses performed in both the DUGL and AGL also were on the same days.

Cells have evolved mechanisms for rapidly adjusting their biochemistry in response to changes in the environment, including radiation21. Most research has focused on the deleterious effects of acute, high or chronic radiation on cells, while some studies have demonstrated a stress response in cells grown at radiation doses that are 10 to 79 times lower than background3. In the present study, V79 cells cultured for 2 days in below-background radiation showed a changed protein profile. A total of 980 proteins were differentially expressed, including 576 proteins that were up-regulated and 404 proteins that were down-regulated, in cells cultured in the DUGL compared to the AGL. Over 70% of the DAPs identified by PRM were consistent with TMT proteomic analysis, implying that TMT proteomic analysis was reliable. These findings suggest protein synthesis was increased in V79 cells cultured in below-background radiation. Consistent with this, TEM of V79 cells cultured in the DUGL showed a hypertrophic ER and obvious Golgi bodies. GO analysis indicated that these DAPs exhibited a wide variety of cellular distributions and functions, which covered metabolic progress and macromolecular binding.

Ribosomal proteins play a critical role in ribosome assembly, protein translation, and cell proliferation. Some sources of extracellular stimulation (e.g. genotoxic chemicals, ionizing or ultraviolet radiation) can result in ribosomal stress and disturb ribosome biogenesis22. In the present study, GO enrichment analysis of DAPs showed that many terms (e.g. ribosome biogenesis, ribosome assembly) involved in ribosome biogenesis were significantly enriched. KEGG pathway analysis also suggested that the DAPs were involved in pathways of ribosome and ribosome biogenesis in eukaryotes. Among the terms enriched in GO and KEGG pathway analysis, ribosomal protein (RP) S3, RPS4, RPS14, RPS15, RPS27, RPL5, RPL6, RPL11, RPL23, RPL26, and RPL37 function to suppress cell proliferation by multiple mechanisms, including p-53ubiquitination and degradation, which leads to cell cycle and proliferation arrest22. In the PPI network, the core proteins were mainly ribosomal proteins, including RPS6, RPS14, RPS16, RPL8, RPL23, RPL3, and RPS18. These findings imply that ribosomal proteins played an essential role in the stress response in V79 cells caused by the deep underground environment and were involved in the multiple mechanisms that led to suppression of cell proliferation and cell survival under the changed environment22.

Spliceosome is a multi-megadalton ribonucleoprotein complex23. Splicing of precursor mRNA catalyzed by the spliceosome is an essential step in eukaryotic gene expression, by which noncoding sequences are removed and coding sequences are ligated together23. The spliceosome is central to the gene expression and protein synthesis required for cell growth and division24. In the present study, 28 DAPs were enriched in the splicesome pathway, and 158 DAPs were enriched in the ribonucleoprotein complex. Most of these proteins were upregulated in cells cultured in the DUGL. These data suggest that reduced background radiation altered gene expression by increasing spliceosome function, which helped V79 cells adapt to the changed environment.

Translation is an essential step in which genetic information is decoded to a functional polypeptide. Eukaryotic translation initiation factors (EIFs) are needed for the initiation phase of eukaryotic translation, helping to stabilize the formation of ribosomal pre-initiation complexes around the start codon, scan mRNA, and locate the initiation codon25. In the present study, 19 EIF protein subunits were up-regulated in V79 cells cultured in the DUGL, 14 of these proteins were involved in the RNA transport pathway and translation. Among those subunits, EIF2 attenuates the rate of translation in eukaryotic cells, allowing cells to conserve resources and initiate adaptive gene expression to restore cellular homeostasis26, and EIF3 can act as both a repressor and activator of translation. As stress proteins are controlled at the translational level27, upregulation of EFIs in response to low background radiation may allow selective translation of mRNAs to maintain the expression of stress proteins, while general protein synthesis is compromised.

Nucleic acid binding has a role in translation regulation. In the present study, GO enrichment analysis of DAPs showed nucleic binding proteins were significantly enriched. RNA-binding motif protein 3(RBM3) is a member of the glycine rich RNA-binding protein family that is induced by cold shock and low oxygen tension. RBM3 expression is essential for proper cell cycle progression and mitosis28. Cold-inducible RNA-binding protein (CIRP) helps cells to adapt to novel environmental conditions, such as UV radiation, by stabilizing specific mRNAs and facilitating their translation29. Both RBM3 and CIRP expression were increased in the cells cultured in the DUGL and indicated that these RNA-binding proteins might play some role in the stress of reduced background radiation.

The ER is a vital organelle with multiple functions, including protein synthesis and folding17. The ER can perceive and transduce environmental signals. ER stress activates the unfolded protein response (UPR), which leads to changes in key mediators of cell survival30. Recent research suggests that ionizing radiation can induce ER stress and initiate the UPR31. In the present study, 23/27 proteins enriched in the protein processing in ER pathway were down regulated in cells cultured in the DUGL. These included ER resident protein 29 (ERp29), protein disulfide isomerase A4 (PDIA4), endoplasmic reticulum chaperone BiP (BiP), also known as glucose-regulated protein 78 kDa (GRP78), and DNAJ homolog subfamily C member 3 (DNAJC3). ERp29 and PDIA4 are up-regulated in response to ER stress. GRP78 is an important molecular chaperone that prevents the aggregation of misfolded proteins in the ER31,32. DNAJC3 is a co-chaperone of GRP78 that attenuates general protein synthesis under ER stress33. This revealed that ER also involved in the stress of reduced background radiation. However, the most proteins down regulated in the cultures in DUGL need to be elucidated in future research.

Mitochondria play an essential role in cellular processes by producing ATP34 . Mitochondria are also involved in stress responses, and mitochondrial morphology reflects the energetic state and viability of cells35. Various environmental factors can affect mitochondrial morphology and metabolic activities (e.g. oxidative phosphorylation and programmed cell death), including laser or exogenous ROS-induced damage, which causes mitochondrial swelling36. In the present study, V79 cells cultured in the DUGL showed mitochondrial swelling, GO analysis revealed proteins enriched in the mitochondrial respiratory chain were dysregulated, and KEGG analysis of the DAPs showed the OXPPL pathway was significantly enriched. OXPPL is an important metabolic pathway that provides energy for cell growth and reproduction37. In V79 cells cultured in below-background radiation, 12/20 proteins enriched in the OXPPL pathway were down-regulated. This potentially altered energy homeostasis in V79 cells and their ability to proliferate. Consistent with these findings, Castillo et al. reported down-regulation of an ATPase in S. oneidensis cultured in low background radiation21.

Environmental stress induces the accumulation of reactive oxygen species (ROS) in cells as a host defense mechanism; however, ROS can cause oxidative stress if produced in excess38. In the present study, GO analysis showed enrichment of proteins involved in oxidoreductase activity and the oxidation–reduction process. This suggested that below-background radiation might induce oxidative stress. Consistent with this, Castillo et al.6 showed that Shewanella oneidensis cultured in low background radiation suffered oxidative stress, activated the SOS response (katB and recA) and up-regulated a putative metal efflux pump (SOA0154).

Our study has some limitations. First, we were unable to measure the levels of cosmic radiation in the DUGL at the CJEM and in the incubators. Second, one batch V79 cells were only maintained in the deep underground environment for a week and the experiments were conducted by a single research team. Longer term experiments investigating different phases of cell growth are required. Third, validation of differential expression of proteins in V79 cells cultured under low background radiation by knockdown and over expression studies should be conducted. Fourth, as ventilation in a deep mine is challenging, radiation was the only environmental factor that could be maintained at a constant level. Last, we expect that environmental factors other than below background radiation influenced V79 cell growth, but these remain to be elucidated.

In conclusion, proliferation of V79 cells was inhibited in the deep underground environment, likely because cells were exposed to reduced background radiation. There were apparent changes in the proteome profile of V79 cells cultured in the DUGL, which affected proteins related to the ribosome, RNA transport, translation, energy, metabolism, and gene spliceosome. These proteins may have induced cellular changes that delayed proliferation but enhanced survival, making cells adaptable to the changing environmental conditions. Our findings provide insight into the cellular stress response that is triggered in the absence of normal levels of radiation.

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