Core level ionization or excitation and Auger relaxation induce clustered DNA damage.

Ionizing radiation causes various types of DNA damage, such as single- (SSBs) and double-strand breaks (DSBs), nucleobase lesions, abasic sites (AP sites), and cross-linking between complementary strands of DNA or DNA and proteins. DSBs are among the most harmful type of DNA damage, inducing serious genetic effects such as cell lethality and mutation. Nucleobase lesions and AP sites, on the other hand, may be less deleterious and are promptly repaired by base excision repair (BER) pathways. Recently, biochemical approaches to quantify nucleobase lesions and AP sites have revealed certain types of non-strand break lesions as harmful DNA damage, called clustered DNA damage. Such clusters can retard nucleobase excision repair enzymes, and can sometimes be converted to DSBs by BER catalysis. This unique character of clustered DNA damage strongly depends on the spatial density of ionization or excitation events occurring at the track end of initial radiation or low energy secondary electrons. In particular, the photoelectric effect of elements comprising biological molecules, followed by emission of Auger electrons, are key factors in determining the future fate of each clustered damage site. This chapter describes biological studies of clustered nucleobase lesions with SSBs or AP sites, and mechanistical studies on core level excitation and Auger relaxation giving rise to clustered DNA damage.

Expression of an X-Ray Irradiated EGFP-Expressing Plasmid Transfected into Nonirradiated Human Cells.

To investigate the repairability of X-ray induced DNA damage, particularly non-double-strand breaks in living cells, enhanced green fluorescent protein (EGFP)-expressing plasmids X-ray irradiated and then transfected into nonirradiated human cells, MCF7 and MCF10A. Live-cell imaging of EGFP fluorescence was performed to measure the efficiency of plasmid repair in cells. The number of EGFP-expressing cells significantly decreased with increasing X-ray dose for both cell lines. The obtained kinetic curves of EGFP expression indicating plasmid repair were quantitatively compared against algebraically calculated ones based on the values of the transfected plasmids that had been treated with nicking or restriction enzymes. Then, assuming a Poisson distribution of single-strand breaks (SSBs), the number of cells carrying these nicked plasmids that could express EGFP were estimated. Our experimental results revealed considerably fewer cells expressing EGFP compared to the expected values we had calculated. These results suggest that the lower proportion of cells expressing EGFP as a measure of plasmid repair was due not only to the complex chemical structures of termini created by SSBs compared to those created by enzyme treatments, but also that base lesions or AP sites proximately arising at the strand-break termini might compromise EGFP expression. These results emphasize that radiation-induced DNA breaks are less repairable than enzymatically induced DNA breaks, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA.

The DsbA-L gene is associated with respiratory function of the elderly via its adiponectin multimeric or antioxidant properties.

Oxidative stress and inflammation play a key role in the age-related decline in the respiratory function. Adipokine in relation to the metabolic and inflammatory systems is attracting growing interest in the field of respiratory dysfunction. The present clinical and experimental studies investigated the role of the disulfide bond-forming oxidoreductase A-like protein (DsbA-L) gene, which has antioxidant and adiponectin multimeric (i.e. activation) properties, on the respiratory function of the elderly. We performed a retrospective longitudinal genotype-phenotype relationship analysis of 318 Japanese relatively elderly participants (mean age ± standard deviation: 67.0 ± 5.8 years) during a health screening program and an in vitro DsbA-L knock-down evaluation using 16HBE14o-cells, a commonly evaluated human airway epithelial cell line. The DsbA-L rs1917760 polymorphism was associated with a reduction in the ratio of forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) and %FEV1 and with the elevation of the prevalence of FEV1/FVC < 70%. We also confirmed that the polymorphism was associated with a decreased respiratory function in relation to a decrease in the ratio of high-molecular-weight adiponectin/total adiponectin (as a marker of adiponectin multimerization) and an increase in the oxidized human serum albumin (as an oxidative stress marker). Furthermore, we clarified that DsbA-L knock-down induced oxidative stress and up-regulated the mucus production in human airway epithelial cells. These findings suggest that the DsbA-L gene may play a role in protecting the respiratory function of the elderly, possibly via increased systemic adiponectin functions secreted from adipocytes or through systemic and/or local pulmonary antioxidant properties.

Melinjo seed extract increases adiponectin multimerization in physiological and pathological conditions.

Melinjo seed extract (MSE) contains large amounts of polyphenols, including dimers of trans-resveratrol (e.g. gnetin C, L, gnemonoside A, B and D), and has been shown to potentially improve obesity. However, there is no clinical evidence regarding the anti-obesity effects of MSE, and its mechanisms are also unclear. We investigated the hypothesis that MSE supplementation increases the adiponectin (APN) multimerization via the up-regulation of disulfide bond A oxidoreductase-like protein (DsbA-L) under either or both physiological and obese conditions. To investigate the effect of MSE on the physiological condition, 42 healthy young volunteers were enrolled in a randomized, double-blind placebo-controlled clinical trial for 14 days. The participants were randomly assigned to the MSE 150 mg/day, MSE 300 mg/day or placebo groups. Furthermore, in order to investigate the effect of MSE on APN levels under obese conditions, we administered MSE powder (500 or 1000 mg/kg/day) to control-diet- or high-fat-diet (HFD)-fed C57BL/6 mice for 4 weeks. All participants completed the clinical trial. The administration of MSE 300 mg/day was associated with an increase in the ratio of HMW/total APN in relation to the genes regulating APN multimerization, including DsbA-L. Furthermore, this effect of MSE was more pronounced in carriers of the DsbA-L rs191776 G/T or T/T genotype than in others. In addition, the administration of MSE to HFD mice suppressed their metabolic abnormalities (i.e. weight gain, increased blood glucose level and fat mass accumulation) and increased the levels of total and HMW APN in serum and the mRNA levels of ADIPOQ and DsbA-L in adipose tissue. The present study suggests that MSE may exert beneficial effects via APN multimerization in relation to the induction of DsbA-L under both physiological and obese conditions.

A Pilot Study Assessing the Possible Combined Effect of Physical Activity and PNPLA3 rs738409 Polymorphism on the Risk for Non-Alcoholic Fatty Liver Disease in the Japanese Elderly General Population.

PURPOSE: The patatin-like phospholipase domain containing protein 3 (PNPLA3) rs738409 polymorphism (c.444C>G) is the most well-known genetic risk factor for non-alcoholic fatty liver disease (NAFLD), but whether or not physical activity influences the association between the PNPLA3 genotype and risk of NAFLD is unclear. PATIENTS AND METHODS: A retrospective longitudinal analysis was conducted among 352 Japanese subjects. Each type of physical activity was assigned a metabolic equivalent (MET), and the subjects were classified into sedentary, low or high groups using the "METS*T" (METs × hours per week) value of 5 or 21 as a threshold. RESULTS: Among the PNPLA3 G/G genotype carriers, the high and low METS*T groups had a lower risk of NAFLD than the sedentary METS*T group (odds ratio [95% confidence interval]: 0.14 [0.02-0.99] and 0.16 [0.03-1.04], respectively). Furthermore, the PNPLA3 C/C or C/G genotype carriers showed no significant difference in the risk of NAFLD among the three METS*T groups. CONCLUSION: The PNPLA3 rs738409 genotype may be associated with the beneficial effects of physical activity on the risk of NAFLD among elderly Japanese individuals. Further comprehensive investigations are therefore needed to verify the preliminary results.

VISUALIZATION OF THE DNA REPAIR PROCESS IN MAMMALIAN CELLS TRANSFECTED WITH EGFP-EXPRESSING PLASMID DNA AFTER EXPOSURE TO X-RAYS IN VITRO.

To investigate the repair process of DNA damage induced by ionizing radiation in isolation from various types of cytoplasmic damage, we transfected X-irradiated enhanced green fluorescent protein (EGFP)-expressing plasmid DNA into non-irradiated mammalian cells using lipofectamine. The repair kinetics of the irradiated plasmids in the cells were visualized under microscopy as the EGFP fluorescence emitted by transfected cells. Using an agarose gel electrophoresis method, the yields of single- and double-strand breaks of the plasmids were also quantified. As positive control experiments, plasmid DNA with single- or double-strand breaks induced by a nicking or restriction enzyme were also transfected into the cells. The DNA repair rates for X-ray-irradiated plasmids were significantly lower than those of the enzymatically digested positive control samples. These results indicate that X-rays could induce less repairable damage than that induced by enzymes.