Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells
<p>Cell recovery and cell viability after 24 h incubation in rotating-wall vessels (RWVs): (<b>A</b>) Change in the initial cell concentration with incubation time at a rotation speed of 10 rpm (6 independent experiments); (<b>B</b>) cell recovery of nontreated and treated cells over experimental conditions before and after 24 h incubation in RWVs rotating at 8.5 rpm (18 independent experiments); and (<b>C</b>) the percentage of live nontreated cells that remained alive after 24 h incubation rotating at a speed of 8.5 rpm (20 independent experiments).</p> "> Figure 2
<p>Apoptosis rate (calculated as indicated in <a href="#ijms-19-03689-t001" class="html-table">Table 1</a>) and residual DNA strand breaks after 24 h incubation in RWVs in µg or 1<span class="html-italic">g</span>. Apoptosis rate is represented by the mean transformed values (<b>A</b>) and (<b>B</b>) and DNA strand breaks are means expressed in Gy dose equivalent (<b>C</b>). Nontreated cells (C), isoproterenol (Iso) and radiation (R) treated cells. Error bars represent +1 SEM. Statistical analysis are summarized in <a href="#ijms-19-03689-t001" class="html-table">Table 1</a>.</p> "> Figure 3
<p>Radiation effects on gene expression relative to non-irradiated cells incubated in (<b>A)</b> 1<span class="html-italic">g</span> and (<b>B</b>) µg. Cells were irradiated either with 0.8 Gy (light bars) or 2 Gy (dark bars) and immediately incubated in 1<span class="html-italic">g</span> (grey bars) or µg (blue bars) for 24 h. Error bars represent SEM. Asterisks represent significant differences in gene expression in cells irradiated with 0.8 Gy compared to cells irradiated with 2 Gy. Statistical method: Krieger. <span class="html-italic">p</span>-value threshold: 0.018 after controlling the FDR (false-discovery rate—see statistical methods) to 5%.</p> "> Figure 4
<p>Cytokine concentration in cell culture medium. Cells were irradiated 2 Gy and subsequently incubated in 1g or µg. After 24 h cytokine concentration was measured. Radiation induced cytokine production in µg but not in 1<span class="html-italic">g</span>. Isoproterenol treatment prior to radiation prevented the production of all cytokines. Bars represent mean + 1 SEM from 10 independent experiments. The synergistic effect of isoproterenol and radiation in µg (Iso × R(µg)) was significant for all four cytokines. Statistical analyses are summarized in <a href="#ijms-19-03689-t004" class="html-table">Table 4</a> and <a href="#ijms-19-03689-t005" class="html-table">Table 5</a>.</p> "> Figure 5
<p>Association between changes in cytokines and corresponding changes in gene expression over the eight experimental conditions for the group of samples irradiated with 2 Gy. BAX, CASP3, PCNA, LIG4, and MDM2 gene expressions were positively associated, while AKT1, TP53, PARP1, OGG1, and APXE1 were negatively associated with cytokines.</p> "> Figure 6
<p>Schematic representation of the treatment conditions. Cells were distributed in eight RCCSVs. Isoproterenol treatment (Iso) and radiation exposure (R) were performed separately and in combination (Iso + R). For the combined treatment, cells were irradiated for 2 min immediately after 2 min isoproterenol (Iso) treatment. Vessels were placed in an incubator and rotated at 8.5 rpm on a vertical (Earth gravity = 1<span class="html-italic">g</span>) or on a horizontal axis (simulated microgravity = µg). Vessels with nontreated cells (C) were incubated in 1<span class="html-italic">g</span> and in µg. After 24 h cells were recovered from the vessels and analyses were performed (DSB = DNA strand breaks).</p> ">
Abstract
:1. Introduction
1.1. Exogenous Factors Affecting DNA Damage Response (DDR)
1.2. Endogenous Factors Affecting DNA Damage Response
2. Results
2.1. Optimization of the Cell Concentration and Rotation Speed in Rotatory Wall Vessels (RWVs)
2.2. Synergistic Effect of Gravity, Radiation and Isoproterenol on Apoptosis
2.3. Effects of Radiation, Microgravity, and Isoproterenol on Gene Expression
2.4. Synergistic Effects of Gravity, Radiation, and Isoproterenol on Cytokine Release
3. Discussion
4. Materials and Methods
4.1. Isolation of PBMCs from Whole Blood
4.2. Experimental Design
4.3. Gene Expression
4.4. Apoptosis
4.5. Detection of DNA Strand Breaks
4.6. Cytokine Quantification
4.7. Statistical Analysis
4.8. Comparison of Cytokine and Gene Expression Data
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Effect | Diff | p Value |
---|---|---|
Apoptosis 0.8 Gy | ||
R (1g) | 0.13 | 0.0000138 # |
µg | −0.1 | 0.0156 |
Iso × g | −0.1 | 0.0202 |
R (µg) | 0.13 | 0.0498 |
Iso (1g) | 0.06 | 0.103 |
Iso (µg) | −0.05 | 0.149 |
Iso × R × g | 0.06 | 0.278 |
Iso (1g, R) | 0.04 | 0.313 |
Iso × R (µg) | 0.04 | 0.34 |
Iso × R (1g) | −0.02 | 0.684 |
Iso (µg, R) | −0.01 | 0.724 |
R × g | 0 | 0.941 |
Apoptosis 2 Gy | ||
R (µg) | 0.37 | 5.32 × 10−15 # |
R (1g) | 0.39 | 0.0000189 # |
Iso × R (1g) | −0.2 | 0.0000635 # |
Iso (1g) | 0.11 | 0.000782 # |
Iso × g | −0.12 | 0.00092 # |
Iso (1g, R) | −0.09 | 0.00276 # |
Iso (µg, R) | −0.06 | 0.00955 |
Iso × R × g | 0.15 | 0.0148 |
µg | −0.07 | 0.0156 |
Iso × R (µg) | −0.05 | 0.123 |
Iso (µg) | −0.01 | 0.218 |
R × g | −0.02 | 0.612 |
DNA strand breaks 2 Gy | ||
µg | 0.78 | 0.000 # |
Iso (1g, R) | 0.50 | 0.000 # |
Iso (µg) | −0.60 | 0.000 # |
Iso × R (µg) | −0.37 | 0.007 # |
R (1g) | 0.35 | 0.010 # |
R (µg) | 0.30 | 0.024 |
Iso (1g) | −0.18 | 0.167 |
Iso × R (1g) | 0.15 | 0.259 |
Iso (µg, R) | −0.07 | 0.607 |
Gene | Effect | Diff | Se | Df | p Value |
---|---|---|---|---|---|
ADRB2 | R (µg) | 0.3327 | 0.0715 | 36.7 | 0.0000417 |
ADRB2 | Iso (1 g) | 0.3107 | 0.0781 | 58.4 | 0.000195 |
BAX | R (µg) | 1.4426 | 0.1567 | 9.69 | 4.22 × 10−6 |
BAX | R (1g) | 1.2942 | 0.1659 | 6.76 | 0.000128 |
CASP3 | Iso (µg) | 0.3497 | 0.094 | 20.5 | 0.0013 |
CASP3 | R (µg) | 0.435 | 0.1185 | 15.2 | 0.00224 |
CASP3 | Iso × R (µg) | −0.4005 | 0.1342 | 61.3 | 0.00408 |
LIG4 | R × g | 0.2239 | 0.0713 | 58.6 | 0.00264 |
LIG4 | Iso (1g, R) | −0.3771 | 0.0976 | 10.2 | 0.00302 |
LIG4 | Iso × R (1g) | −0.308 | 0.0989 | 16.3 | 0.00655 |
LIG4 | Iso (µg, R) | −0.3202 | 0.1 | 12.5 | 0.00722 |
MDM2 | R (1g) | 0.8083 | 0.0571 | 67.4 | 7.40 × 10−22 |
MDM2 | R (µg) | 0.8957 | 0.1039 | 12.9 | 1.01 × 10−6 |
MDM2 | Iso (1g) | −0.1626 | 0.0553 | 16.3 | 0.00938 |
OGG1 | Iso (1g) | −0.22 | 0.0722 | 49.6 | 0.00368 |
PARP1 | R (1g) | −0.1836 | 0.0468 | 44.9 | 0.000295 |
PARP1 | µg | −0.2918 | 0.0809 | 21.6 | 0.0016 |
PARP1 | Iso (1g, R) | −0.129 | 0.0427 | 64.3 | 0.00365 |
PARP1 | Iso (µg, R) | −0.1946 | 0.0647 | 25.4 | 0.00584 |
PARP1 | Iso (1g) | −0.1066 | 0.0409 | 91.6 | 0.0107 |
PCNA | R (1g) | 0.6586 | 0.0666 | 55.3 | 7.80 × 10−14 |
PCNA | R (µg) | 0.8327 | 0.1228 | 9.58 | 0.00006 |
PTEN | Iso (1g, R) | −0.1878 | 0.0446 | 18.5 | 0.000494 |
PTEN | Iso × R (µg) | −0.3164 | 0.0717 | 9.44 | 0.00151 |
PTEN | Iso × g | 0.2721 | 0.0893 | 46.3 | 0.0038 |
PTEN | Iso (µg) | 0.179 | 0.059 | 35.4 | 0.00453 |
TP53 | R (1g) | −0.349 | 0.0851 | 22.2 | 0.000466 |
XRCC5 | Iso (1g, R) | −0.3624 | 0.0402 | 26.2 | 1.62 × 10−9 |
XRCC5 | Iso × R (µg) | −02794 | 0.049 | 30.1 | 3.18 × 10−6 |
XRCC5 | Iso (µg, R) | −0.2963 | 0.0526 | 16.6 | 0.0000326 |
XRCC5 | Iso × R (1g) | −0.3697 | 0.0794 | 23.2 | 0.000108 |
Gene | Effect | Diff | Se | Df | p Value |
---|---|---|---|---|---|
ADRB2 | R × g | −0.3369 | 0.0992 | 34.4 | 0.00174 |
ADRB2 | R (µg) | 0.481 | 0.0848 | 34.4 | 2.20 × 10−6 |
ADRB2 | Iso (1g) | 0.5047 | 0.1009 | 33.7 | 0.0000175 |
ADRB2 | Iso (µg, R) | 0.5138 | 0.1528 | 19.1 | 0.00324 |
ADRB2 | Iso × R × g | 0.6219 | 0.2297 | 48.1 | 0.00935 |
ADRB2 | R (1g) | 0.8179 | 0.1239 | 19.2 | 2.42 × 10−6 |
AKT1 | R (1g) | −0.2202 | 0.0598 | 18.9 | 0.00158 |
AKT1 | R (µg) | −0.1696 | 0.0509 | 29.3 | 0.00234 |
APEX1 | R (µg) | −0.4306 | 0.054 | 52.7 | 1.27 × 10−10 |
BAX | R (1g) | 1.9229 | 0.102 | 5.96 | 1.54 × 10−6 |
BAX | R (µg) | 1.9254 | 0.0481 | 28.2 | 1.97 × 10−26 |
CASP3 | Iso (µg, R) | −0.4001 | 0.0794 | 55.4 | 5.38 × 10−6 |
MDM2 | R (µg) | 1.6218 | 0.0694 | 9.65 | 8.14 × 10−10 |
MDM2 | R (1g) | 1.6469 | 0.1196 | 7.75 | 1.00 × 10−6 |
PARP1 | R (µg) | −0.3947 | 0.0465 | 32.5 | 9.41 × 10−10 |
PARP1 | R (1g) | −0.2701 | 0.0602 | 20.8 | 0.000208 |
PCNA | R (1g) | 1.2425 | 0.0831 | 13.2 | 1.19 × 10−9 |
PCNA | R (µg) | 1.3845 | 0.046 | 80 | 0 |
TP53 | R (µg) | −0.6312 | 0.056 | 47.6 | 5.04 × 10−15 |
TP53 | R (1g) | −0.4132 | 0.0749 | 25.7 | 8.95 × 10−6 |
XRCC5 | Iso (µg, R) | −0.2727 | 0.0225 | 146 | 7.45 × 10−24 |
XRCC5 | Iso × R (µg) | −0.2678 | 0.0538 | 30.4 | 0.000024 |
Cyt | Effect | Diff | Se | Df | p Value |
---|---|---|---|---|---|
GM-CSF | R (µg) | −0.3165 | 0.0939 | 15.5 | 0.00404 |
IL-10 | R (µg) | 0.9723 | 0.3034 | 23 | 0.00393 |
IL-12p70 | Iso (µg, R) | −2 | 0.3567 | 30.7 | 3.87 × 10-6 |
IL-12p70 | Iso (µg) | −2.6 | 0.4945 | 108 | 7.38 × 10-7 |
IL-12p70 | R (µg) | 2.3 | 0.5767 | 32.6 | 0.000354 |
IL-1B | R (µg) | 1.309 | 0.3461 | 8.67 | 0.00463 |
IL-1B | R × g | 1.4022 | 0.4936 | 117 | 0.00531 |
IL-2 | R (µg) | 0.1426 | 0.0432 | 65.4 | 0.00156 |
IL-4 | R (µg) | 0.0851 | 0.0262 | 42.4 | 0.00223 |
IL-5 | Iso (µg) | 0.1321 | 0.0407 | 105 | 0.00155 |
IL-6 | Iso (µg, R) | −2 | 0.4747 | 11.2 | 0.0014 |
IL-6 | Iso (µg) | −2 | 0.5868 | 26.8 | 0.00208 |
IL-6 | R (µg) | 2 | 0.4385 | 11.8 | 0.000685 |
TNFα | µg | −0.0355 | 0.0098 | 26.1 | 0.00118 |
TNFα | Iso (µg, R) | 0.0359 | 0.007 | 97.8 | 1.41 × 10-6 |
TNFα | Iso (µg) | 0.0402 | 0.0047 | 542 | 7.43 × 10-17 |
TNFα | Iso (1g, R) | 0.0675 | 0.0189 | 14.7 | 0.0029 |
TNFα | R (µg) | −0.028 | 0.0069 | 13.3 | 0.0013 |
Cyt | Effect | Diff | Se | Df | p Value |
---|---|---|---|---|---|
GM-CSF | Iso (µg, R) | −4.4 | 0.5784 | 31.2 | 1.36 × 10−8 |
GM-CSF | Iso (1g) | −2.8 | 0.624 | 24.5 | 0.000147 |
GM-CSF | Iso × R (µg) | −2.95 | 0.8635 | 43.9 | 0.00138 |
GM-CSF | Iso × R × g | −6.5 | 1.5547 | 32.9 | 0.000202 |
GM-CSF | R (µg) | 2.85 | 0.4337 | 16.1 | 6.28 × 10−6 |
GM-CSF | R × g | 4.15 | 0.9974 | 18.5 | 0.000556 |
IL-10 | Iso (µg, R) | −3.05 | 0.6062 | 7.7 | 0.00114 |
IL-10 | Iso (µg) | −1.5 | 0.4324 | 36.2 | 0.00137 |
IL-10 | R (µg) | 2.9 | 0.3257 | 19.9 | 2.26 × 10−8 |
IL-12p70 | Iso (µg, R) | 0.2468 | 0.0496 | 8.39 | 0.000941 |
IL-12p70 | Iso × R (µg) | 0.171 | 0.049 | 15.4 | 0.0032 |
IL-12p70 | R (µg) | −0.2024 | 0.0416 | 18.8 | 0.000111 |
IL-13 | Iso (µg, R) | −3.55 | 0.4825 | 90.5 | 8.18 × 10−11 |
IL-13 | Iso (1g) | −2.8 | 0.5063 | 68.6 | 5.42 × 10−7 |
IL-1B | Iso (µg, R) | −2.4713 | 0.5444 | 13.6 | 0.000493 |
IL-1B | Iso (1g) | −0.9541 | 0.214 | 73.4 | 0.0000292 |
IL-1B | Iso × R (µg) | −2.1307 | 0.5551 | 12.8 | 0.0021 |
IL-1B | R (µg) | 2.709 | 0.4346 | 11.7 | 0.0000485 |
IL-2 | µg | −0.1161 | 0.0403 | 106 | 0.00475 |
IL-2 | Iso (µg. R) | −0.2931 | 0.0611 | 20.6 | 0.000103 |
IL-2 | Iso (1g) | −0.1642 | 0.0402 | 48 | 0.000164 |
IL-2 | R (µg) | 0.2543 | 0.0444 | 51.2 | 5.46 × 10−7 |
IL-2 | R × g | 0.3121 | 0.0776 | 22.8 | 0.000542 |
IL-4 | Iso (µg, R) | −0.1665 | 0.0475 | 12 | 0.00432 |
IL-4 | Iso (µg) | −0.0993 | 0.0326 | 29.2 | 0.00488 |
IL-4 | R (µg) | 0.1426 | 0.0414 | 44.9 | 0.00124 |
IL-5 | Iso (µg, R) | −4.4 | 0.5475 | 29.3 | 6.85 × 10−9 |
IL-5 | Iso (1g) | −3.1 | 0.7242 | 29.5 | 0.000181 |
IL-5 | Iso × R × g | −5.9 | 1.5538 | 23 | 0.000932 |
IL-5 | R (µg) | 2.4 | 0.5134 | 60.8 | 0.0000168 |
IL-6 | Iso (µg, R) | −3.7 | 0.7387 | 8.3 | 0.000933 |
IL-6 | Iso (µg) | −2.3 | 0.3979 | 240 | 2.30 × 10−8 |
IL-6 | Iso (1g) | −3.4 | 0.587 | 11.1 | 0.000115 |
IL-6 | R (µg) | 2.3 | 0.3138 | 41.5 | 5.26 × 10−9 |
IL-7 | µg | 0.0751 | 0.0154 | 73.1 | 6.50 × 10−6 |
IL-7 | Iso (µg, R) | 0.1521 | 0.0246 | 23.2 | 2.47 × 10−6 |
IL-7 | Iso (1g) | 0.1062 | 0.0216 | 23.3 | 0.0000547 |
IL-7 | Iso × g | −0.0889 | 0.023 | 30.5 | 0.000532 |
IL-7 | Iso × R (µg) | 0.1348 | 0.0289 | 23.2 | 0.000108 |
IL-7 | Iso × R × g | 0.2149 | 0.0417 | 20.5 | 0.0000453 |
IL-7 | R (µg) | −0.161 | 0.0216 | 30.3 | 2.47 × 10−8 |
IL-7 | R × g | −0.1724 | 0.0254 | 36.2 | 6.06 × 10−8 |
IL-8 | Iso (µg, R) | −3.2 | 0.5387 | 21.8 | 5.82 × 10−6 |
IL-8 | R (µg) | 2.5 | 0.6377 | 53.7 | 0.000252 |
TNFα | Iso (µg, R) | −4 | 0.4773 | 16.4 | 2.48 × 10−7 |
TNFα | Iso (µg) | −2.9 | 0.511 | 26.6 | 5.24 × 10−6 |
TNFα | Iso (1g) | −4.2 | 0.5913 | 24.1 | 2.38 × 10−7 |
TNFα | R (µg) | 2.7 | 0.4469 | 22.6 | 3.96 × 10−6 |
TNFα | R × g | 2.8 | 0.693 | 10.2 | 0.00227 |
Cellular Parameters | Nr. of Experiments Performed | Statistical Model | Dependent Variable | Multiple Testing | |
---|---|---|---|---|---|
0.8 Gy | 2 Gy | ||||
Apoptosis and DNA strand breaks | 15 | 16 | Mixed model regression. Random effects at sample level | 2 sin−1 √p where p is the proportion of cells undergoing apoptosis | FWER control to 5% (12 tests) |
-- | 6 | Mixed model regression. Random effects at sample level. | equivalent dose (Gy) | FWER control to 5% (9 tests) | |
Gene expression | 9 | 10 | Mixed model regression. Random effects at PCR-plate level. | log2 cycles normalized to average values of three housekeeping genes | FDR control to 5% (180 tests) |
Cytokines | 10 | 10 | Mixed model regression. Random effects at sample level. | log concentration | FDR control to 5% (0.8 Gy) control to 1% (2 Gy) (144 tests) |
Analysis Effects (Contrasts) | Description |
---|---|
R (µg) | Effect of radiation alone in simulated microgravity |
R (1g) | Effect of radiation alone in Earth gravity |
Iso (µg) | Effect of isoproterenol alone in simulated microgravity |
Iso (1g) | Effect of isoproterenol alone in Earth gravity |
Iso (µg, R) | Effect of isoproterenol on irradiated cells in simulated microgravity |
Iso (1g, R) | Effect of isoproterenol on irradiated cells in Earth gravity |
Iso × R (µg) | Synergistic effect of Isoproterenol and radiation in simulated microgravity |
Iso × R (1g) | Synergistic effect of Isoproterenol and radiation in Earth gravity |
µg | Average effect of microgravity over all combinations of the other factors (R and Iso) |
Iso × g | Average synergistic effect of Isoproterenol and gravity for non-irradiated cells |
R × g | Average synergistic effect of radiation and gravity for cells without Isoproterenol |
Iso × R × g | Three-way synergistic effect of Isoproterenol, radiation, and gravity |
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Moreno-Villanueva, M.; Feiveson, A.H.; Krieger, S.; Kay Brinda, A.; Von Scheven, G.; Bürkle, A.; Crucian, B.; Wu, H. Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells. Int. J. Mol. Sci. 2018, 19, 3689. https://doi.org/10.3390/ijms19113689
Moreno-Villanueva M, Feiveson AH, Krieger S, Kay Brinda A, Von Scheven G, Bürkle A, Crucian B, Wu H. Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells. International Journal of Molecular Sciences. 2018; 19(11):3689. https://doi.org/10.3390/ijms19113689
Chicago/Turabian StyleMoreno-Villanueva, Maria, Alan H. Feiveson, Stephanie Krieger, AnneMarie Kay Brinda, Gudrun Von Scheven, Alexander Bürkle, Brian Crucian, and Honglu Wu. 2018. "Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells" International Journal of Molecular Sciences 19, no. 11: 3689. https://doi.org/10.3390/ijms19113689