Intracellular reactive oxygen species level in blood cells of atomic bomb survivors is increased due to aging and radiation exposure.

Although reactive oxygen species (ROS) play important roles in immune responses, excessive ROS production and accumulation might enhance the risk of inflammation-related diseases. Moreover, impaired immune function and the acceleration of pre-clinically persistent inflammation due to aging and radiation exposure have been observed in atomic bomb (A-bomb) survivors more than 60 years post-exposure. Meanwhile, the effects of aging and radiation exposure on ROS production in immune cells have not been characterized. This study investigated the relationship between intracellular ROS (H2O2 and O2•-) levels in blood cells or T cell subsets and serum iron, ferritin, and C-reactive protein (CRP) levels, as well as how these variables are affected by age and radiation exposure in A-bomb survivors. We examined 2495 Hiroshima A-bomb survivors. Multiple linear regression models adjusted for confounding factors indicated that intracellular O2•- levels in monocytes, granulocytes, and lymphocytes, and particularly in memory CD8+ T cells, including effector memory and terminally differentiated effector memory CD8+ T cells, increased with radiation dose. Additionally, serum iron, ferritin, and CRP levels affected intracellular ROS levels in specific blood cell types and T cell subsets. Serum CRP levels increased significantly with increasing age and radiation dose. Finally, when divided into three groups according to serum CRP levels, dose-dependent increases in the intracellular O2•- levels in blood cells and central memory and effector memory CD8+ T cells were most prominently observed in the high-CRP group. These results suggest that an increase in the levels of certain intracellular ROS, particularly after radiation exposure, might be linked to enhanced inflammatory status, including elevated serum CRP levels and reduced serum iron levels. This study reveals that aging and radiation exposure increase oxidative stress in blood cells, which is involved in impaired immune function and accelerated pre-clinically persistent inflammation in radiation-exposed individuals.

Impact of early life exposure to ionizing radiation on influenza vaccine response in an elderly Japanese cohort.

The objective of this study was to evaluate effects of whole body radiation exposure early in life on influenza vaccination immune responses much later in life. A total of 292 volunteers recruited from the cohort members of ongoing Adult Health Study (AHS) of Japanese atomic bomb (A-bomb) survivors completed this observational study spanning two influenza seasons (2011-2012 and 2012-2013). Peripheral blood samples were collected prior to and three weeks after vaccination. Serum hemagglutination inhibition (HAI) antibody titers were measured as well as concentrations of 25 cytokines and chemokines in culture supernatant from peripheral blood mononuclear cells, with and without in vitro stimulation with influenza vaccine. We found that influenza vaccination modestly enhanced serum HAI titers in this unique cohort of elderly subjects, with seroprotection ranging from 18 to 48% for specific antigen/season combinations. Twelve percent of subjects were seroprotected against all three vaccine antigens post-vaccination. Males were generally more likely to be seroprotected for one or more antigens post-vaccination, with no differences in vaccine responses based on age at vaccination or radiation exposure in early life. These results show that early life exposure to ionizing radiation does not prevent responses of elderly A-bomb survivors to seasonal influenza vaccine.

Pentanoic acid induces thymic stromal lymphopoietin production through Gq/11 and Rho-associated protein kinase signaling pathway in keratinocytes.

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic skin inflammation. Short-chain fatty acids (SCFAs), including pentanoic acid, are products of bacterial metabolism and are associated with allergic skin disorders. However, whether SCFAs induce TSLP production is still unclear. In this study, we evaluated the effect of SCFAs on TSLP production and found that pentanoic acid was the most efficacious of the tested SCFAs. The Gq/11 inhibitor YM-254890 and the Rho-associated protein kinase (ROCK) inhibitor Y-27632 inhibited pentanoic acid-induced TSLP production, as did transfection with Gq/11 siRNA. These results suggested that pentanoic acid-induced TSLP production was mediated by Gq/11 and ROCK, providing insights into a novel TSLP production pathway in keratinocytes. The novel mechanism of TSLP production is expected to support the development of TSLP-regulating approaches in allergic skin disorders.

Long term effects of radiation exposure on telomere lengths of leukocytes and its associated biomarkers among atomic-bomb survivors.

Ionizing radiation (IR) is a major source of cellular damage and the immediate cellular response to IR has been well characterized. But the long-term impact of IR on cell function and its relationship with aging are not known. Here, we examined the IR effects on telomere length and other biomarkers 50 to 68 years post-exposure (two time points per person) in survivors of the atomic bombing at Hiroshima during WWII. We found that telomere length of leukocytes was inversely correlated with the dose of IR (p=0.008), and this effect was primarily found in survivors who were exposed at younger ages; specifically those <12 years old (p=0.0004). Although a dose-related retardation of telomere shortening with age was observed in the cross-sectional data, longitudinal follow-up after 11 years did not show IR exposure-related alteration of the rate of telomere shortening with age. In addition, IR diminished the associations between telomere length and selected aging biomarkers that were observed in survivors with no dose. These included uric acid metabolism, cytokines, and blood T cell counts. These findings showed long-lasting detrimental effects of IR on telomere length of leukocytes in both dose- and age-at-exposure dependent manner, and on alterations of biomarkers with aging.

CD14 and IL18 gene polymorphisms associated with colorectal cancer subsite risks among atomic bomb survivors.

Colorectal cancer (CRC) is a common malignancy worldwide, and chronic inflammation is a risk factor for CRC. In this study, we carried out a cohort study among the Japanese atomic bomb (A-bomb) survivor population to investigate any association between immune- and inflammation-related gene polymorphisms and CRC. We examined the effects of six single-nucleotide polymorphisms of CD14 and IL18 on relative risks (RRs) of CRC. Results showed that RRs of CRC, overall and by anatomic subsite, significantly increased with increasing radiation dose. The CD14-911A/A genotype showed statistically significant higher risks for all CRC and distal CRC compared with the other two genotypes. In addition, the IL18-137 G/G genotype showed statistically significant higher risks for proximal colon cancer compared with the other two genotypes. In phenotype-genotype analyses, the CD14-911A/A genotype presented significantly higher levels of membrane and soluble CD14 compared with the other two genotypes, and the IL18-137 G/G genotype tended to be lower levels of plasma interleukin (IL)-18 compared with the other two genotypes. These results suggest the potential involvement of a CD14-mediated inflammatory response in the development of distal CRC and an IL18-mediated inflammatory response in the development of proximal colon cancer among A-bomb survivors.

Effects of IL-10 haplotype and atomic bomb radiation exposure on gastric cancer risk.

Gastric cancer (GC) is one of the cancers that reveal increased risk of mortality and incidence in atomic bomb survivors. The incidence of gastric cancer in the Life Span Study cohort of the Radiation Effects Research Foundation (RERF) increased with radiation dose (gender-averaged excess relative risk per Gy = 0.28) and remains high more than 65 years after exposure. To assess a possible role of gene-environment interaction, we examined the dose response for gastric cancer incidence based on immunosuppression-related IL-10 genotype, in a cohort study with 200 cancer cases (93 intestinal, 96 diffuse and 11 other types) among 4,690 atomic bomb survivors participating in an immunological substudy. Using a single haplotype block composed of four haplotype-tagging SNPs (comprising the major haplotype allele IL-10-ATTA and the minor haplotype allele IL-10-GGCG, which are categorized by IL-10 polymorphisms at -819A>G and -592T>G, +1177T>C and +1589A>G), multiplicative and additive models for joint effects of radiation and this IL-10 haplotyping were examined. The IL-10 minor haplotype allele(s) was a risk factor for intestinal type gastric cancer but not for diffuse type gastric cancer. Radiation was not associated with intestinal type gastric cancer. In diffuse type gastric cancer, the haplotype-specific excess relative risk (ERR) for radiation was statistically significant only in the major homozygote category of IL-10 (ERR = 0.46/Gy, P = 0.037), whereas estimated ERR for radiation with the minor IL-10 homozygotes was close to 0 and nonsignificant. Thus, the minor IL-10 haplotype might act to reduce the radiation related risk of diffuse-type gastric cancer. The results suggest that this IL-10 haplotyping might be involved in development of radiation-associated gastric cancer of the diffuse type, and that IL-10 haplotypes may explain individual differences in the radiation-related risk of gastric cancer.

Evaluation of systemic markers of inflammation in atomic-bomb survivors with special reference to radiation and age effects.

Past exposure to atomic bomb (A-bomb) radiation has exerted various long-lasting deleterious effects on the health of survivors. Some of these effects are seen even after >60 yr. In this study, we evaluated the subclinical inflammatory status of 442 A-bomb survivors, in terms of 8 inflammation-related cytokines or markers, comprised of plasma levels of reactive oxygen species (ROS), interleukin (IL)-6, tumor necrosis factor α (TNF-α), C-reactive protein (CRP), IL-4, IL-10, and immunoglobulins, and erythrocyte sedimentation rate (ESR). The effects of past radiation exposure and natural aging on these markers were individually assessed and compared. Next, to assess the biologically significant relationship between inflammation and radiation exposure or aging, which was masked by the interrelationship of those cytokines/markers, we used multivariate statistical analyses and evaluated the systemic markers of inflammation as scores being calculated by linear combinations of selected cytokines and markers. Our results indicate that a linear combination of ROS, IL-6, CRP, and ESR generated a score that was the most indicative of inflammation and revealed clear dependences on radiation dose and aging that were found to be statistically significant. The results suggest that collectively, radiation exposure, in conjunction with natural aging, may enhance the persistent inflammatory status of A-bomb survivors.

Caspase-independent cell death without generation of reactive oxygen species in irradiated MOLT-4 human leukemia cells.

To improve our understanding of ionizing radiation effects on immune cells, we investigated steps leading to radiation-induced cell death in MOLT-4, a thymus-derived human leukemia cell. After exposure of MOLT-4 cells to 4 Gy of X-rays, irradiated cells sequentially showed increase in intracellular reactive oxygen species (ROS), decrease in mitochondrial membrane potential, and eventually apoptotic cell death. In the presence of the caspase inhibitor z-VAD-fmk, irradiated cells exhibited necrotic characteristics such as mitochondrial swelling instead of apoptosis. ROS generation was not detected during this necrotic cell death process. These results indicate that radiation-induced apoptosis in MOLT-4 cells requires elevation of intracellular ROS as well as activation of a series of caspases, whereas the cryptic necrosis program--which is independent of intracellular ROS generation and caspase activation--is activated when the apoptosis pathway is blocked.

High-throughput spectrophotometric assay of reactive oxygen species in serum.

The derivatives of reactive oxygen metabolites (D-ROM) test has been developed to determine the amount of oxygen-centered free radicals in a blood sample as a marker of oxidative stress. This study aims to improve the D-ROM test and develop an automated assay system by use of a clinical chemistry analyzer. Five microliters of serum was added to 1 well of a 96-well microtiter plate for a total 240microl of reaction solution containing alkylamine and metals. This was followed by automatic mixing, incubation and measurement of reactive oxygen species (ROS) levels as a color development at 505nm using a spectrophotometer with catalytic capability for transition metals. This assay system was used to measure serum levels of ROS in cigarette smokers and never-smokers, by way of example. The levels of serum ROS determined by this system correlate with the amounts of free radicals and peroxides, which reacted with various molecules in the body and formed stable metabolites. This test can use frozen sera as well as fresh ones. The inter- and intra-deviation of this system was within 5% and showed consistent linearity in the range between 4 and 500mg/l of hydrogen peroxides. Serum ROS levels among smokers increased with the number of cigarettes smoked per day (36.5% increment per pack per day; P<0.0001). This assay system will be a simple, inexpensive, and reliable tool for assessing oxidative stress in human populations. Our preliminary results on cigarette smoking imply that this assay system has potential for application in various epidemiological and clinical settings.

Identification of the NKG2D haplotypes associated with natural cytotoxic activity of peripheral blood lymphocytes and cancer immunosurveillance.

We have previously shown that natural cytotoxic activity of peripheral blood lymphocytes was inversely related to cancer development based on a prospective cohort study. The genetic fraction of cytotoxic activity needs to be clarified, identifying individuals immunogenetically susceptible to cancer. A case-control study within the cohort members was designed: 102 cancer cases with peripheral lymphocyte DNA available and three control groups, each of which consisted of 204 subjects with each tertile level of cytotoxic activity. We first compared two control groups with high and low cytotoxic activity in terms of the single nucleotide polymorphisms in the natural killer complex gene region on chromosome 12p, identifying the haplotype alleles that were associated with the activity. Next, cancer risks were assessed for these haplotypes. We found two haplotype blocks, each of which generated two major haplotype alleles: low-activity-related LNK1 (frequency 0.478 and 0.615 in groups with high and low activity, respectively; P < 0.00008) and high-activity-related HNK1 (0.480 and 0.348; P < 0.0001), LNK2 (0.711 and 0.821; P < 0.0002), and HNK2 (0.272 and 0.174; P < 0.0008). These NKG2D haplotype alleles showed a significant difference between cases (0.632 for LNK1 and 0.333 for HNK1) and controls (0.554 for LNK1 and 0.406 for HNK1). The haplotype HNK1/HNK1 revealed a decreased risk of cancer (odds ratio, 0.471; 95% confidence interval, 0.233-0.952) compared with LNK1/LNK1. Individuals who are genetically predisposed to have low or high natural cytotoxic activity can in part be determined by NKG2D haplotyping, which in turn reveals an increased or decreased risk of cancer development.

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH.

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.

HLA haplotype is associated with diabetes among atomic bomb survivors.

We examined radiation effects on the relationship between diabetes development and genetic background in atomic-bomb (A-bomb) survivors. Our main aim in this study was to shed light on the role of genetic background in diabetes onset among A-bomb survivors by studying possible relationships between human leukocyte antigen (HLA) genotypes and the diabetes in patients and controls. We examined the effects of different HLA haplotypes on type 2 diabetes development by determining the DQA1 and DRB1 alleles of Hiroshima A-bomb survivors (111 diabetic patients and 774 controls) using the polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method. We noted an increased risk of diabetes in the higher dose group among these patients (trend p = 0.001). The risk of the most heavily exposed group was significantly higher than that of the unexposed group or the low-dose group especially in survivors with the DQA1*03-DRB1*09 or DQA1*0401-DRB1*08 haplotypes (trend p = 0.002 or p = 0.05, respectively). By contrast, in people with other haplotypes, the risk did not increase significantly with increasing dose. These results suggest that individuals with specific HLA haplotypes may have an increased risk of diabetes with increased-dose categories.