Exposure of keratinocytes to non­thermal dielectric barrier discharge plasma increases the level of 8­oxoguanine via inhibition of its repair enzyme.

Oxidative stress enhances cellular DNA oxidation and may cause mutations in DNA bases, including 8­oxoguanine (8­oxoG). Our recent study reported that exposure of cells to non­thermal dielectric barrier discharge (DBD) plasma generates reactive oxygen species and damages DNA. The present study investigated the effect of non­thermal DBD plasma exposure on the formation of 8­oxoG in HaCaT human keratinocytes. Cells exposed to DBD plasma exhibited increased level of 8­oxoG. In addition, mRNA and protein expression levels of 8­oxoguanine glycosylase 1 (OGG1), an 8­oxoG repair enzyme, were reduced in plasma­exposed cells. Furthermore, the expression level of nuclear factor erythroid 2­related factor 2 (Nrf2), a transcription factor that regulates OGG1 gene expression, was reduced following exposure to DBD plasma. Pretreatment of cells with an antioxidant, N­acetyl cysteine (NAC), prior to plasma exposure suppressed the formation of 8­oxoG and restored the expression levels of OGG1 and Nrf2. In addition, phosphorylation of protein kinase B (Akt), which regulates the activation of Nrf2, was reduced following plasma exposure. However, phosphorylation was restored by pretreatment with NAC. These findings suggested that non­thermal DBD plasma exposure generates 8­oxoG via inhibition of the Akt­Nrf2­OGG1 signaling pathway in HaCaT cells.

Effects of high voltage nanosecond pulsed plasma and micro DBD plasma on seed germination, growth development and physiological activities in spinach.

In this study, we analyzed seed germination, seedling growth, and physiological aspects after treatment with high voltage nanosecond pulsed plasma and micro DBD plasma in spinach (Spinacia oleracea L.), a green leafy vegetable known to have low germination rate. Both germination and dry weight of seedlings increased after high voltage pulse shots were applied to spinach seeds. However seeds treated with many shots (10 shots) showed a decrease in germination rate and seedling growth. Seeds treated with air DBD plasma exhibited slightly higher germination and subsequent seedling growth than those treated with N2 plasma. Seed surface was degenerated after treated with high voltage pulsed plasma and micro DBD plasma but no significant difference in the degree of degeneration was observed among micro DBD plasma treatment time. Level of GA3 hormone and mRNA expression of an amylolytic enzyme-related gene in seeds were elevated 1 day after treatment with high voltage pulsed plasma. The relative amount of chlorophyll and total polyphenols in spinach seedlings grown from seeds treated with air DBD plasma was increased in 30 s, 1 min, and 3 min treatments. Taken together, our results suggest a possibility that plasma can enhance seed germination by triggering biochemical processes in seeds.

Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress.

The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components.

Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death.

Side-by-side comparison of DNA damage induced by low-energy electrons and high-energy photons with solid TpTpT trinucleotide.

The genotoxic effects of high-energy ionizing radiation have been largely attributed to the ionization of H2O leading to hydroxyl radicals and the ionization of DNA leading mostly to damage through base radical cations. However, the contribution of low-energy electrons (LEEs; ≤ 10 eV), which involves subionization events, has been considered to be less important than that of hydroxyl radicals and base radical cations. Here, we compare the ability of LEEs and high-energy X-ray photons to induce DNA damage using dried thin films of TpTpT trinucleotide as a simple and representative model for DNA damage. The main radiation-induced damage of TpTpT as measured by high-performance liquid chromatography (HPLC) with UV detection and HPLC coupled to tandem mass spectrometry analyses included thymine release (-Thy), strand breaks (pT, Tp, pTpT, TpTp, and TpT), and the formation of base modifications [5,6-dihydrothymine (5,6-dhT), 5-hydroxymethyluracil (5-hmU), and 5-formyluracil (5-fU)]. The global profile of products was very similar for both types of radiation indicating converging pathways of formation. The percent damage of thymine release, fragmentation, and base modification was 20, 19, and 61 for high-energy X-rays, respectively, compared to 35, 13, and 51 for LEEs (10 eV). Base release was significantly lower for X-rays. In both cases, phosphodiester bond cleavage gave mononucleotides (pT and Tp) and dinucleotides (pTpT and TpTp) containing a terminal phosphate as the major fragments. For base modifications, the ratio of reductive (5,6-dhT) to oxidative products (5-hmU plus 5-fU) was 0.9 for high-energy X-rays compared to 1.7 for LEEs. These results indicate that LEEs give a similar profile of products compared to ionizing radiation.

Radiation-induced formation of 2',3'-dideoxyribonucleosides in DNA: a potential signature of low-energy electrons.

We have identified a series of modifications of the 2'-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. The modifications consist of 2',3'-dideoxyribonucleoside derivatives of T, C, A, and G, as identified by enzymatic digestion and LC-MS/MS. Under dry conditions, the yield of these products was 6- to 44-fold lower than the yield of 8-oxo-7,8-dihydroguanine. We propose that 2',3'-dideoxyribonucleosides are generated from the reaction of low-energy electrons with DNA, leading to cleavage of the C3'-O bond and formation of the corresponding C3'-deoxyribose radical.

Fundamental mechanisms of DNA radiosensitization: damage induced by low-energy electrons in brominated oligonucleotide trimers.

The replacement of nucleobases with brominated analogs enhances DNA radiosensitivity. We examine the chemistry of low-energy electrons (LEEs) in this sensitization process by experiments with thin films of the oligonucleotide trimers TBrXT, where BrX = 5-BrU (5-bromouracil), 5-BrC (5-bromocytosine), 8-BrA (8-bromoadenine), or 8-BrG (8-bromoguanine). The products induced from irradiation of thin (∼ 2.5 nm) oligonucleotide films, with 10 eV electrons, under ultrahigh vacuum (UHV) are analyzed by HPLC-UV. The number of damaged brominated trimers ranges from about 12 to 15 × 10(-3) molecules per incident electron, whereas under the identical conditions, these numbers drop to 4-7 × 10(-3) for the same, but nonbrominated oligonucleotides. The results of HPLC analysis show that the main degradation pathway of trinucleotides containing brominated bases involve debromination (i.e., loss of the bromine atom and its replacement with a hydrogen atom). The electron-induced sum of products upon bromination increases by factors of 2.1 for the pyrimidines and 3.2 for the purines. Thus, substitution of any native nucleobase with a brominated one in simple models of DNA increases LEE-induced damage to DNA and hence its radiosensitivity. Furthermore, besides the brominated pyrimidines that have already been tested in clinical trials, brominated purines not only appear to be promising sensitizers for radiotherapy, but could provide a higher degree of radiosensitization.

Effect of low-energy electron irradiation on DNA damage by Fe3+ ion.

We investigated the combined effects of low-energy electron irradiation and Fe(3+) ion on DNA damage. We used lyophilized pBR322 plasmid DNA films with various concentrations (0 ~ 7 mM) of Fe(3+) ions and irradiation with monochromatic, low-energy 3 or 5 eV electrons for these studies. DNA-Fe(3+) films were recovered and analyzed by agarose gel electrophoresis to identify and compare the effects of Fe(3+) ions and/or low-energy electrons alone or in combination on DNA damage. In nonirradiated DNA-Fe(3+) films, there was little DNA damage observed (less than 10% of the total DNA loaded on the gel appeared damaged) for Fe(3+) ion up to 7 mM concentration. In irradiated DNA films without Fe(3+) ions, there was also very little DNA damage observed (less than 3% of the total DNA loaded on the gel appeared damaged). However, when DNA-Fe(3+) films, were irradiated with low-energy electrons, DNA damage was significantly increased compared to the sum of the damage caused both by either Fe(3+) ion or low-energy electrons irradiation alone. We proposed that both DEA and/or electron transfer processes might play a role in the enhanced DNA damage when DNA-Fe(3+) films were irradiated by low-energy electrons.

DNA damage induced by low-energy electrons: conversion of thymine to 5,6-dihydrothymine in the oligonucleotide trimer TpTpT.

Low-energy electrons (LEE) induce single- and double-strand breaks in DNA. To investigate the mechanism of LEE-induced DNA damage, nucleotides and short oligonucleotide were irradiated with monoenergetic electrons in the solid state and the modifications were observed by chemical analyses. With 10 eV electrons and TpTpT as the target, approximately one-third of the total damage of TpTpT involves cleavage of the phosphodiester-sugar bond (C-O) and the N-glycosidic bond (C-N). Here we focus on the remaining two-thirds of the damage. The major products were observed to elute between TpT and TpTpT on the HPLC chromatogram. Of these products, three modifications were identified as XpTpT, TpXpT and TpTpX, where X  =  5,6-dihydrothymine, on the basis of comparison with standard compounds using HPLC and mass spectrometry. These results suggest that 5,6-dihydrothymine is a major product of the reaction of LEE with DNA.