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The effects of residual radiation from atomic bombs have been considered to be minimal because of its low levels of external radioactivity. However, studies involving atomic bomb survivors exposed to only residual radiation in Hiroshima and Nagasaki have indicated possible adverse health effects. Thus, we investigated the biological effects of radioactive dust of manganese dioxide 56 ( 56 MnO 2 ), a major radioisotope formed in soil by neutron beams from a bomb. Previously, we investigated C57BL mice exposed to 56 MnO 2 and found pulmonary gene expression changes despite low radiation doses. In this study, we examined the effects in a radiation-sensitive strain of mice, BALB/c, and compared them with those in C57BL mice. The animals were exposed to 56 MnO 2 particles at two radioactivity levels and examined 3 and 65 days after exposure. The mRNA expression of pulmonary pathophysiology markers, including Aqp1, Aqp5, and Smad7, and radiation-sensitive genes, including Bax, Phlda3, and Faim3, was determined in the lungs. The radiation doses absorbed in the lungs ranged from 110 to 380 mGy; no significant difference was observed between the two strains. No exposure-related pathological changes were observed in the lungs of any group. However, the mRNA expression of Aqp1 was significantly elevated in C57BL mice but not in BALB/c mice 65 days after exposure, whereas no changes were observed in external γ-rays (2 Gy) in either strain. In contrast, Faim3, a radiation-dependently downregulated gene, was reduced by 56 MnO 2 exposure in BALB/c mice but not in C57BL mice. These data demonstrate that inhalation exposure to 56 MnO 2 affected the expression of pulmonary genes at doses <380 mGy, which is comparable to 2 Gy of external γ-irradiation, whereas the responses differed between the two mouse strains.

The investigation of the radiation effects of the atomic bombing in Hiroshima and Nagasaki has revealed concerns about the impact of the residual radioactive dust produced in the soil. Manganese-56 is one of the major radioisotopes produced by neutrons from the bomb; hence, we previously examined the biological effects of manganese dioxide-56 ( 56 MnO 2 ) in Wistar rats, in which significant changes were found in the lung. In the present study, ten-week-old male C57BL mice were exposed to three doses of radioactive 56 MnO 2 , stable MnO 2 particles, or external γ-rays (2 Gy) to further examine the effects of 56 MnO 2 in a different species. The estimated absorbed radiation doses from 56 MnO 2 were 26, 96, and 250 mGy in the lung. The animals were examined at 3, 14, and 70 days post exposure. Histologically, no exposure-related changes were found in the lungs of any group. However, pulmonary mRNA expression of aquaporin 1, which is a useful marker for lung pathophysiology, was significantly elevated at 14 and 70 days, although no such changes were found in the mice exposed to external γ-rays (2 Gy). These data indicated that the inhalation exposure to 56 MnO 2 particles, with <250 mGy of organ doses, produced significant biological responses in the lung.

We have studied the biological effects of the internal exposure to radioactive manganese-56 dioxide ( 56 MnO 2 ), the major radioisotope dust found in soil after atomic bomb explosions. Our previous study of blood chemistry indicated a possible adverse effect of 56 MnO 2 on the liver. In the present study, we further examined the effects on the liver by determining changes in hepatic gene expressions. Male Wistar rats were exposed to 56 MnO 2 particles (three groups with the whole-body doses of 41, 90, and 100 mGy), stable MnO 2 particles, or external 60 Co γ-rays (2 Gy), and were examined together with the non-treated control group on postexposure day 3 and day 61. No histopathological changes were observed in the liver. The mRNA expression of a p53-related gene, the cyclin-dependent kinase inhibitor 1A, increased in 56 MnO 2 as well as in γ-ray irradiated groups on postexposure day 3 and day 61. The expression of a stress-responsive gene, nuclear factor κB, was also increased by 56 MnO 2 and γ-rays on postexposure day 3. However, the expression of cytokine genes (interleukin-6 or chemokine ligand 2) or fibrosis-related TGF-β/Smad genes ( Tgfb1 , Smad3 , or Smad4 ) was not altered by the exposure. Our data demonstrated that the internal exposure to 56 MnO 2 particles at less than 0.1 Gy significantly affected the short-term gene expressions in the liver in a similar manner with 2 Gy of external γ-irradiation. These changes may be adaptive responses because no changes occurred in cytokine or TGF-β/Smad gene expressions.

To understand the radiation effects of the atomic bombing of Hiroshima and Nagasaki among the survivors, radiation from neutron-induced radioisotopes in soil should be considered in addition to the initial radiation directly received from the bombs. 56 Mn, which emits both β particles and γ-rays, is one of the dominant radioisotopes created in soil by neutrons from the bomb. Thus we investigated the biological effects of internal exposure to 56 MnO 2 particle in the lung of male Wistar rats comparing to the effects of external 60 Co-γ irradiation. Absorbed doses of internal irradiation of lungs were between 25 and 65 mGy in 56 MnO 2 -exposed animals, while the whole body doses were between 41 and 100 mGy. Animals were examined on days 3 and 61 after the exposure. There were no remarkable pathological changes related to 56 MnO 2 particle exposure. However, mRNA and protein expressions of aquaporin 5 increased significantly in the lung tissue on day 3 postexposure in 56 MnO 2 groups (by 1.6 and 2.9 times, respectively, in the highest dose group). Smad7 mRNA expression was also significantly elevated by 30% in the highest dose group of 56 MnO 2 . Our data demonstrated that internal exposure to 56 MnO 2 induced significant biological responses including gene expression changes in the lungs, while external 60 Co-γ irradiation of 2 Gy did not show any changes.

The experiment was performed in support of a Japanese initiative to investigate the biological effects of irradiation from residual neutron-activated radioactivity that resulted from the A-bombing. Radionuclide 56 Mn (T 1/2  = 2.58 h) is one of the main neutron-activated emitters during the first hours after neutron activation of soil dust particles. In our previous studies (2016-2017) related to irradiation of male Wistar rats after dispersion of 56 MnO 2 powder, the internal doses in rats were found to be very inhomogeneous: distribution of doses among different organs ranged from 1.3 Gy in small intestine to less than 0.0015 Gy in some of the other organs. Internal doses in the lungs ranged from 0.03 to 0.1 Gy. The essential pathological changes were found in lung tissue of rats despite a low level of irradiation. In the present study, the dosimetry investigations were extended: internal doses in experimental mice and rats were estimated for various activity levels of dispersed neutron-activated 56 MnO 2 powder. The following findings were noted: (a) internal radiation doses in mice were several times higher in comparison with rats under similar conditions of exposure to 56 MnO 2 powder. (b) When 2.74 × 10 8  Bq of 56 MnO 2 powder was dispersed over mice, doses of internal irradiation ranged from 0.81 to 4.5 Gy in the gastrointestinal tract (small intestine, stomach, large intestine), from 0.096 to 0.14 Gy in lungs, and doses in skin and eyes ranged from 0.29 to 0.42 Gy and from 0.12 to 0.16 Gy, respectively. Internal radiation doses in other organs of mice were much lower. (c) Internal radiation doses were significantly lower in organs of rats with the same activity of exposure to 56 MnO 2 powder (2.74 × 10 8  Bq): 0.09, 0.17, 0.29, and 0.025 Gy in stomach, small intestine, large intestine, and lungs, respectively. (d) Doses of internal irradiation in organs of rats and mice were two to four times higher when they were exposed to 8.0 × 10 8  Bq of 56 MnO 2 (in comparison with exposure to 2.74 × 10 8  Bq of 56 MnO 2 ). (e) Internal radiation doses in organs of mice were 7-14 times lower with the lowest 56 MnO 2 amount (8.0 × 10 7  Bq) in comparison with the highest amount, 8.0 × 10 8  Bq, of dispersed 56 MnO 2 powder. The data obtained will be used for interpretation of biological effects in experimental mice and rats that result from dispersion of various levels of neutron-activated 56 MnO 2 powder, which is the subject of separate studies.

To investigate the biological effects of internal exposure of radioactive 56 MnO 2 powder, the major radioisotope dust in the soil after atomic bomb explosions, on male reproductive function, the gene expression of the testes and the prostate was examined. Ten-week-old male Wistar rats were exposed to three doses of radioactive 56 MnO 2 powder (41-100 mGy in whole body doses), stable MnO 2 powder, or external 60 Co γ-rays (2 Gy). Animals were necropsied on Days 3 and 61 postexposure. The mRNA expressions of testicular marker protein genes and prostatic secretory protein genes were quantified by Q-RT-PCR. On Day 3 postexposure, the testicular gene expressions of steroidogenesis-related enzymes, Cyp17a1 and Hsd3b1, decreased in 56 MnO 2 -exposed groups. Germ cell-specific Spag4 and Zpbp mRNA levels were also reduced. On postexposure Day 61, the Cyp11a1 gene expression became significantly reduced in the testes in the group exposed to the highest dose of 56 MnO 2 , while another steroidogenesis-related StAR gene mRNA level reduced in the 60 Co γ-rays group. There were no differences in Spag4 and Zpbp mRNA levels among groups on Day 61. No histopathological changes were observed in the testes in any group following exposure. Expression in the prostatic protein genes, including CRP1, KS3, and PSP94, significantly decreased in 56 MnO 2 -exposed groups as well as in the 60 Co γ-rays group on Day 61 postexposure. These data suggest that the internal exposure to 56 MnO 2 powder, at doses of less than 100 mGy, affected the gene expressions in the testis and the prostate, while 2 Gy of external γ-irradiation was less effective.

Internal radiation exposure from neutron-induced radioisotopes environmentally activated following atomic bombing or nuclear accidents should be considered for a complete picture of pathologic effects on survivors. Inhaled hot particles expose neighboring tissues to locally ultra-high doses of β-rays and can cause pathologic damage. 55 MnO 2 powder was activated by a nuclear reactor to make 56 MnO 2 which emits β-rays. Internal exposures were compared with external γ-rays. Male Wistar rats were administered activated powder by inhalation. Lung samples were observed by histological staining at six hours, three days, 14 days, two months, six months and eight months after the exposure. Synchrotron radiation - X-ray fluorescence - X-ray absorption near-edge structure (SR-XRF-XANES) was utilized for the chemical analysis of the activated 56 Mn embedded in lung tissues. 56 Mn beta energy spectrum around the particles was calculated to assess the local dose rate and accumulated dose. Hot particles located in the bronchiole and in damaged alveolar tissue were identified as accumulations of Mn and iron. Histological changes showed evidence of emphysema, hemorrhage and severe inflammation from six hours through eight months. Apoptosis was observed in the bronchiole epithelium. Our study shows early event damage from the locally ultra-high internal dose leads to pathogenesis. The trigger of emphysema and hemorrhage was likely early event damage to blood vessels integral to alveolar walls., Competing Interests: The authors declare that they have no conflicts of interest.