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) 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.
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 underground [5–7, 16]. 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 ground[8, 17]. 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 months[8, 17]. Short term stress responses in cells undergoing an acute environmental change differ from the adaptive response seen in cells exposed to chronic stress[18]. 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 ground [8, 17].
We speculate that reduced cosmic ray muons flux 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 radiation [19]. Other environmental parameters, including light, O2 levels, relative humidity, temperature, concentration of CO2, air pressure and terrestrial radiation 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 bar [20, 21]. Air pressure in the DUGL was significantly different from the AGL; however, the difference was within the margin of measurement error. 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.
Cells have evolved mechanisms for rapidly adjusting their biochemistry in response to changes in the environment, including radiation[22]. 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 background[3]. 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. 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 term enrichment analysis of DAPs that were up-regulated in V79 cells cultured in the DUGL revealed enrichment of proteins involved in the ribosome, translation, and nucleotide binding. KEGG pathway analysis showed significant enrichment in the ribosome and RNA transport pathways. Ribosomal proteins play a critical role in ribosome assembly, protein translation, and cell proliferation. Some extracellular stimulations can result in ribosomal stress and disturb ribosome biogenesis[23]. In the present study, a total of 108 ribosomal proteins(RPs), ribosome biogenesis associated proteins, and proteins involved in the ribosome pathway were up-regulated in V79 cells cultured in below-background radiation. GO term enrichment analysis, KEGG pathway analysis [RP S3a (G3HKG8), RP S4 (P47961), RP S14 (P62265), RP S15a (G3IKE2), RPS27 (A0A061IMX1), RP L5 (G3HNJ6), RP L6 (G3GU30), RP L11 (G3HMV2), RP L23 (G3H5W4) RP L26 (G3GY47)] and PPI analysis [RP S23 (G3HIY3), RP S20 (G3I2D3), RPS17 (P63274), RPS27 (A0A061IMX1), RP L5 (G3HNJ6), RP L6 (G3GU30), RP L11(G3HMV2)] implied that these ribosomal proteins were involved in the multiple mechanisms that lead to suppression of cell proliferation and cell cycle arrest, including p53 ubiquitination and degradation[23].
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 codon[24]. In the present study, GO term enrichment analysis and KEGG pathway analysis revealed 7 EIF protein subunits [EIF2α (G3H1M4), EIF3, EIF 1(G3HLU5), EIF 2α, EIF 3 subunit C (A0A061I773) and G (G3H6E1)] were up-regulated in V79 cells cultured in the DUGL, and five of these proteins were involved in the RNA transport pathway. Among these, EIF2α attenuates the rate of translation in eukaryotic cells, allowing cells to conserve resources and initiate adaptive gene expression to restore cellular homeostasis[25], and EIF3 can act as both a repressor and activator of translation. As stress proteins are controlled at the translational level[26], 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. Accordingly, Castillo et al. reported a down-regulation of ribosomal proteins and tRNA genes in Shewanella oneidensis cultures deprived of background levels of radiation in a mine 655 m underground, indicating a marked decrease in general protein translation.[22]
Nucleotide binding proteins have a role in translation regulation. In the present study, nucleotide binding proteins were up-regulated in V79 cells cultured in below-background radiation, including RNA-binding motif protein 3 (RBM3, D5FGC9) and cold-inducible RNA-binding protein (CIRP, P60826). 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 mitosis[27]. CIRP helps cells to adapt to novel environmental conditions, such as UV radiation, by stabilizing specific mRNAs and facilitating their translation[28], 39].
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 excess [29]. In the present study, proteins involved in oxidation-reduction reactions, including oxygen binding, oxygen transporter activity, and hemoglobin subunits (G3ICM8, hemoglobin subunit epsilon-Y2; G3ICM5, hemoglobin subunit beta; G3HBR8, hemoglobin subunit alpha) were up-regulated in V79 cells cultured in the DUGL. Hemoglobin is a major host respiratory protein that can also be specifically activated by pathogens to produce ROS[28]. The hemoglobin subunit epsilon 1 (HBE1) has been implicated in the radiation sensitivity and resistance of colorectal cancer cells [29], and hemoglobin over expression affected O2 homeostasis or suppressed oxidative stress in murine MN9D and SV40-MES13 cells, respectively. Castillo et al.[5] 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). Similarly, V79 cells grown in below-background radiation may increase the transcription of hemoglobin in response to an increase in intracellular ROS.
ROS can induce cellular DNA damage[30]. Base excision repair is an important response to cellular DNA damage caused by free radicals and other reactive species generated by metabolism[31]. Base excision repair can proceed by two pathways, a proliferating cell nuclear antigen (PCNA, P57761)-dependent pathway that utilizes DNA POLδ/ε (long patch repair) or a PCNA-independent pathway that utilizes POLβ (short patch repair) [31, 32]. In the present study, the POLδ (G3I9M7)/ε(G3HHZ8) subunits, PCNA and HMGB1 (a factor that protects cells from injury, P07156), were up-regulated in V79 cells cultured in the DUGL, suggesting that below-background radiation led to cellular DNA damage and preferential induction of the long patch base excision genes.
The ER is a vital organelle with multiple functions, including protein synthesis and folding[18]. 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 survival[33]. Recent research suggests that ionizing radiation can induce ER stress and initiate the UPR[34]. In the present study, ER resident protein 29 (ERp29), protein disulfide isomerase A4 (PDIA4, G3IDT6), endoplasmic reticulum chaperone BiP (BiP, G3I8R9), also known as glucose-regulated protein 78 kDa (GRP78), and DNAJ homolog subfamily C member 3 (DNAJC3, G3H8H7) were down-regulated in V79 cells cultured in below-background radiation. 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 ER[34, 35]. DNAJC3 is a co-chaperone of GRP78 that attenuates general protein synthesis under ER stress[36]. In contrast, peptidyl-prolyl cis-trans isomerase (PPIase) and its subunits were up-regulated in V79 cells cultured in the DUGL. PPIase represents a rate limiting step in protein folding, catalyses cis to trans isomerization of peptidyl prolyl bonds[37, 38] and is involved in many biological processes. PPIase has been implicated in stress tolerance in wheat; therefore, PPIase activity in V79 cells may have been responsible for enhanced protection against decreased background radiation[37].
Mitochondria play an essential role in cellular processes by producing ATP [39]. Mitochondria are also involved in stress responses, and mitochondrial morphology reflects the energetic state and viability[40]. 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 swelling[41]. In the present study, V79 cells cultured in the DUGL showed mitochondrial swelling, proteins involved in the respiratory chain were down-regulated, and KEGG analysis of the DAPs revealed the OXPPL pathway was significantly enriched. OXPPL is an important metabolic pathway that provides energy for cell growth and reproduction[42]. In V79 cells cultured in below-background radiation, the OXPPL and Huntingdon’s disease pathways were down-regulated, including mitochondrial cytochrome oxidase (Q8WBA9, cytochrome c oxidase subunit 2), NADH-ubiquinone oxidoreductase (Q27PP5, NADH-ubiquinone oxidoreductase chain 5; Q27PQ4, NADH-ubiquinone oxidoreductase chain 2) and ATP synthase subunits (G3HL06, ATP synthase subunit g; P14414, ATP synthase protein 8).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 radiation[22].