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. 2023 Nov 7;9(1):26.
doi: 10.1038/s41514-023-00123-3.

Spontaneous p53 activation in middle-aged C57BL/6 mice mitigates the lifespan-extending adaptive response induced by low-dose ionizing radiation

Masaoki Kohzaki  1 , Keiji Suzuki  2 , Akira Ootsuyama  3 , Ryuji Okazaki  4
Affiliations

Spontaneous p53 activation in middle-aged C57BL/6 mice mitigates the lifespan-extending adaptive response induced by low-dose ionizing radiation

Masaoki Kohzaki et al. NPJ Aging. .

Abstract

Understanding the biological effects of low-dose (<100 mGy) ionizing radiation (LDR) is technically challenging. We investigated age-dependent LDR effects using adaptive response experiments in young (7-to 12-week-old) and middle-aged (40-to 62-week-old) C57BL/6 mice. Compared with 3 Gy irradiation, 0.02 Gy preirradiation followed by 3 Gy irradiation prolonged life in young mice but not middle-aged mice. Preirradiation also suppressed irradiation-induced 53BP1 repair foci in the small intestines, splenic apoptosis, and p53 activity in young mice but not middle-aged mice. Young p53+/- C57BL/6 mice did not show these adaptive responses, indicating that insufficient p53 function in young mice mitigated the adaptive responses. Interestingly, p53 activation in middle-aged mice spontaneously became approximately 4.5-fold greater than that in young mice, possibly masking LDR stresses. Furthermore, adaptive responses in young mice, but not in middle-aged mice, suppressed some senescence-associated secretory phenotype (SASP) factors (IL-6, CCL2, CCL5, CXCL1). Thus, LDR-induced adaptive responses associated with specific SASP factors may be attenuated by a combination of reduced DNA damage sensor/transducer function and chronic p53 activation in middle-aged mice.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overview of the experimental process for determining the adaptive response in C57BL/6 mice.
a Four different conditions were set: I. Nonirradiated controls; II. 0.02 Gy (100 µGy/min)-irradiated mice; III. 3 Gy (0.72 Gy/min)-irradiated mice; and IV. 0.02 Gy-preirradiated, 72-h interval, 3 Gy-irradiated mice. The timing of sacrifice for the four groups was the same. Specifically, group IV mice were preirradiated with 0.02 Gy at 3 days before 3 Gy irradiation, and both group III mice and group IV mice were irradiated with 3 Gy on the day of sacrifice. After the last IR exposure, we collected samples at 4 h, 24 h, and 72 h and analyzed them. The experiment at the tissue level in the intestines (blue) was conducted 4 h and 24 h after the final IR exposure. The experiment at the cellular level in the spleen (orange) was conducted 4 h and 72 h after the final IR exposure. The experiment at the intracellular molecular level (purple) was conducted 4 h after the final IR exposure. The time scale is not exactly drawn. The same experiments were conducted in (b) 7 to 12-week-old (young) C57BL/6 mice as experiment 1 and (c) 40 to 62-week-old (middle-aged) C57BL/6 mice as experiment 2. d Comparison of these mice to assess the different biological outcomes at the tissue, cellular, and intracellular molecular levels. These outcomes can be discussed in the context of the lifespan-extending adaptive response induced by very low-dose IR.
Fig. 2
Fig. 2. Kaplan‒Meier survival curves of experiments 1 and 2.
Moribund mice were euthanized via cervical dislocation and necropsied. Gehan‒Breslow‒Wilcoxon tests were used to calculate the significance of the survival curves. Each experiment was conducted several times with different numbers of mice, and the total result is shown. Time 0 indicates the point at which the mouse was born. a Kaplan‒Meier survival curves of the young (7- to 12-week-old) control C57BL/6 mice (n = 42), 0.02 Gy-exposed mice (n = 42), 0.02 Gy + 3 Gy-exposed mice (n = 44) and 3 Gy-exposed mice (n = 41) are shown. The number of cohorts in each group was eight for the control group, four for the 0.02 Gy group, eight for the 3 Gy group, and four for the 0.02 + 3 Gy group. b Kaplan‒Meier survival curves of the middle-aged (40- to 62-week-old) control C57BL/6 mice (n = 25), 0.02 Gy-exposed mice (n = 26), 0.02 Gy + 3 Gy-exposed mice (n = 28) and 3 Gy-exposed mice (n = 26). The number of cohorts in each group was four for the control group, three for the 0.02 Gy group, five for the 3 Gy group, and four for the 0.02 + 3 Gy group.
Fig. 3
Fig. 3. DNA damage kinetics after IR exposure in mouse intestines in experiment 1 and experiment 2.
Scale bars 0.1 mm. a Representative images of 53BP1 foci (red) and DAPI (blue) at 4 h after IR exposure in the crypts of young C57BL/6 mice. The enlarged images are shown in green next to the original images. b Numbers of 53BP1 foci per cell at 4 h after IR exposure in young C57BL/6 mice. Five mice per condition were used, and data were obtained at 10 randomly selected sites. The data show the mean (s.d.). The p values were determined by the two-tailed t test. c Numbers of 53BP1 foci per cell at 24 h after IR in young C57BL/6 mice. Five mice per condition were used, and the data were obtained at 10 randomly selected sites. The data show the mean (s.d.). The p values were determined by the two-tailed t test. d Representative images of 53BP1 foci (red) and DAPI (blue) at 4 h after IR exposure in the crypts of middle-aged C57BL/6 mice. e Comparison of the numbers of 53BP1 foci per cell at 4 h after IR exposure in young and middle-aged C57BL/6 mice. Five mice per condition were used, and the data were obtained at 10 randomly selected sites. The data show the mean (s.d.). The p values were determined by the two-tailed t test. f The numbers of 53BP1 foci per cell at 10 min (immediately) after IR exposure in young C57BL/6 mice. The numbers of 53BP1 foci (red FL) per cell (blue FL) at 10 min after IR 3 Gy and 0.02 + 3 Gy in young C57BL/6 mice are shown. Five mice per condition were used, and the data were obtained at 10 randomly selected sites.
Fig. 4
Fig. 4. Apoptosis induction in splenocytes after IR exposure in experiment 1 and experiment 2.
a Representative images of apoptosis induction (PI negative, Annexin-V positive; dotted squares) in all the examined groups based on FACS analysis. The apoptosis induction ratio at 72 h after 0.02 Gy irradiation was used as a control. We observed continuous apoptosis in young C57BL/6 mice 72 h after 3 Gy. To visually distinguish the adaptive response between 3 Gy and 0.02 + 3 Gy at 4 h after IR, the results for the 3 Gy-exposed group at 4 h after IR group are shown in green. b Percentages of apoptosis induction (PI negative, Annexin-V positive) in young C57BL/6 mice subjected to different treatments (four mice: 72 h after 0.02 Gy, eight mice: 72 h after 3 Gy, five mice: 72 h after 0.02 + 3 Gy, six mice: 4 h after 3 Gy, and four mice: 4 h after 0.02 + 3 Gy). The data show the mean (s.d.). The p values were determined by Welch’s t test or Mann–Whitney U test. c The percentages of apoptosis induction (PI negative, Annexin-V positive) in middle-aged C57BL/6 mice subjected to different treatments (five mice: 72 h after 0.02 Gy, four mice: 72 h after 3 Gy, four mice: 72 h after 0.02 + 3 Gy, six mice: 4 h after 3 Gy, and five mice: 4 h after 0.02 + 3 Gy). The data show the mean (s.d.). The p values were determined by Welch’s t test or Mann–Whitney U test.
Fig. 5
Fig. 5. Quantification of p53 activity in the spleen at 4 h after IR by simple western blotting in experiment 1 and experiment 2.
To avoid individual differences, the same amount of protein was obtained from four independent mice under each condition. a Simple western blotting images of p53 (left) and p53 with serine 15 phosphorylation (Ser15p, right) under four different conditions in young and middle-aged C57BL/6 mice. Simple western blotting images of β-Tubulin as determined using the same samples from four different conditions in young and middle-aged C57BL/6 mice as a loading control. b Quantification of p53 (upper, red) and p53 Ser15p (lower, orange) under four different conditions (control, 0.02 Gy, 3 Gy, and 0.02 + 3 Gy) in young (left) and middle-aged (right) C57BL/6 mice. c Relative p53 activity was calculated as the p53 Ser15p level divided by the total p53 expression level in young (left) and middle-aged (right) C57BL/6 mice. Specifically, we calculated 442/5726 (=0.077), 821/9179 (=0.089), 6598/33351 (=0.198), and 4826/43063 (=0.112) for the control group, 0.02 Gy group, 3 Gy group, 0.02 + 3 Gy group, respectively, in young mice. Then, we normalized to obtain 1 for the control group, 1.16 (0.089/0.077) for the 0.02 Gy group, 2.57 (0.198/0.077) for the 3 Gy group, and 1.45 (0.112/0.077) for the 0.02 + 3 Gy group, to assess the effects of the adaptive response. d As we noticed spontaneous p53 activation in middle-aged C57BL/6 mice, we calculated the relative p53 activation in middle-aged C57BL/6 mice compared with young C57BL/6 mice. Specifically, we calculated 442/5726 (=0.077) and 838/2430 (=0.345) for young and middle-aged control samples, respectively. Then, we calculated 4.48 (0.345/0.077) as the relative activation in middle-aged mice compared with young mice.
Fig. 6
Fig. 6. p53 activity and proteins involved upstream and downstream of the p53 pathway in the spleen, lungs and SI at 4 h post-IR exposure were quantified by standard western blotting in experiment 1.
a Western blotting images of MDM2 (top), p53 Ser15p (second from top), p53 (third from top), PCNA (third from bottom), p21 (second from bottom), and p19ARF (bottom) in young C57BL/6 mice under 3 Gy or 0.02 + 3 Gy conditions. b Quantification of p53 Ser15p divided by p53 under 3 Gy or 0.02 + 3 Gy conditions. Quantification of p19ARF, MDM2, and p21 divided by PCNA under 3 Gy or 0.02 + 3 Gy conditions. The data show the mean (s.d.). The p values were determined by Welch’s t test or Mann–Whitney U test. c Simplified model of the p53 activation pathway. In the p53 upstream pathway, MDM2 can ubiquitinate p53 for degradation by the proteome, which is inhibited by p19ARF. p21 is activated by p53. Arrow: activation; Bar: inhibition. The green arrows indicate the changes in the 0.02 + 3 Gy group compared with the 3 Gy group in the young C57BL/6 mice. d Western blotting images of p53 Ser15p (top: long exposure; middle top: short exposure), p53 (middle bottom), and PCNA (bottom) in four independent samples from aged and young C57BL/6 mice under spontaneous conditions. All blots derive from the same experiment and were processed in parallel. Quantification of p53 Ser15p divided by p53 under spontaneous conditions. The data show the mean (s.d.). The p values were determined by Mann–Whitney U test.
Fig. 7
Fig. 7. Evaluation of p53 function for adaptive responses in young C57BL/6 mice using heterogeneously p53-inactivated C57BL/6 mice.
The experiments were conducted in (a) 7 to 12-week-old young p53+/− C57BL/6 mice as experiment 3. b Kaplan‒Meier survival curves of experiments 3. Gehan‒Breslow‒Wilcoxon tests were used to calculate the significance of the survival curves. Each experiment was conducted several times with different numbers of mice, and the total result is shown. Time 0 indicates the point at which the mouse was born. c Representative images of 53BP1 foci (red) and DAPI (blue) at 4 h after IR exposure in the crypts of young p53+/− C57BL/6 mice. Scale bars 0.1 mm. The enlarged images are shown in green next to the original images. The numbers of 53BP1 foci per cell at 4 h after IR exposure in young p53+/− C57BL/6 mice are shown below the images. Five mice per condition were used, and data were obtained at 10 randomly selected sites. The data show the mean (s.d.). The p values were determined by the two-tailed t test.
Fig. 8
Fig. 8. Relative mRNA expression levels of p53-related genes and SASP genes in the spleen.
All mRNA expression was calculated relative to the expression of housekeeping gene (GAPDH), and the mRNA expression levels in young control mice are shown as 1. Each dot indicates the result for an individual mouse. The data show the mean (s.d.). The p values were determined by Welch’s t test or Mann–Whitney U test. a mRNA expression levels of p53-related factors, with MDM2 and p21 as direct regulators, CCNG1 as a cell cycle factor, and NOXA and PUMA as apoptosis-inducing factors. b mRNA expression levels of the senescence biomarker p16, the hormesis-related transcription factor NRF2, and SASP markers (IL-6, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, MMP3, NF-κB, PAI-1, TNFα, VEGF) along with the NF-κB inhibitor IκB.
Fig. 9
Fig. 9. Graphical summary of the findings obtained in this study.
a The reduced DNA damage at the tissue level (blue) and the subsequently reduced requirement of the DNA repair machinery at the intracellular molecular level (purple) may reflect adaptation to worse DNA damage in young C57BL/6 mice. In addition, continuous elimination of damaged cells by apoptosis at the cellular level in the tissue (orange) occurs in young C57BL/6 mice. Moreover, during the adaptive response in young mice, the mRNA levels of some SASP factors, such as IL-6, CCL2, and CXCL1, are decreased, while the mRNA levels of IκB, which is essential for NFκB suppression, are increased (boxed figure). Combined, these responses result in lifespan extension after exposure to very low doses of IR in young C57BL/6 mice (right). b Worse DNA damage at the tissue level (blue) but subsequent exhaustion of the DNA repair machinery at the intracellular molecular level (purple) may reflect much less adaptation to worse DNA damage in middle-aged C57BL/6 mice than in young C57BL/6 mice. Continuous elimination of damaged cells by apoptosis at the cellular level (orange) is not observed in middle-aged C57BL/6 mice. Combined, these results suggest that there is no lifespan extension induced by very low doses of IR in middle-aged C57BL/6 mice as a consequence of the chronic inflammation of unrepaired cells (right).
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