Hair Follicle Damage after 100 mGy Low-Dose Fractionated X-Ray Irradiation and the Protective Effects of TEMPOL, a Stable Nitroxide Radical, against Radiation.

The effects of long-term low-dose X-ray irradiation on the outer root sheath (ORS) cells of C3H/He mice were investigated. Mice were irradiated with a regime of 100 mGy/day, 5 days/week, for 12 weeks (Group X) and the results obtained were compared to those in a non-irradiated control (Group C). Potential protection against ORS cells damage induced by this exposure was investigated by adding the stable nitroxide radical 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) at 1 mM to the drinking water of mice (Group X + TEMPOL). The results obtained were compared with Group C and a non-irradiated group treated with TEMPOL (Group C + TEMPOL). After fractionated X-ray irradiation, skin was removed and ORS cells were examined by hematoxylin and eosin staining and electron microscopy for an abnormal nuclear morphology and nuclear condensation changes. Fractionated X-irradiated mice had an increased number of ORS cells with an abnormal nuclear morphology as well as nuclear condensation changes. Sections were also immunohistochemically examined for the presence of TdT-mediated dUTP nick-end labeling (TUNEL), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), vascular endothelial growth factor (VEGF), nitrotyrosine, heme oxygenase 1 (HO-1), and protein gene product 9.5 (PGP 9.5). Significant increases were observed in TUNEL, 8-OHdG, and 4-HNE levels in ORS cells from mice in Group X. Electron microscopy also showed irregular shrunken ORS cells in Group X. These changes were prevented by the presence of TEMPOL in the drinking water of the irradiated mice. TEMPOL alone had no significant effects. These results suggest that fractionated doses of radiation induced oxidative damage in ORS cells; however, TEMPOL provided protection against this damage, possibly as a result of the rapid reaction of this nitroxide radical with the reactive oxidants generated by fractionated X-ray irradiation.

Mitochondria Play Essential Roles in Intracellular Protection against Oxidative Stress-Which Molecules among the ROS Generated in the Mitochondria Can Escape the Mitochondria and Contribute to Signal Activation in Cytosol?

Questions about which reactive oxygen species (ROS) or reactive nitrogen species (RNS) can escape from the mitochondria and activate signals must be addressed. In this study, two parameters, the calculated dipole moment (debye, D) and permeability coefficient (Pm) (cm s-1), are listed for hydrogen peroxide (H2O2), hydroxyl radical (•OH), superoxide (O2•-), hydroperoxyl radical (HO2•), nitric oxide (•NO), nitrogen dioxide (•NO2), peroxynitrite (ONOO-), and peroxynitrous acid (ONOOH) in comparison to those for water (H2O). O2•- is generated from the mitochondrial electron transport chain (ETC), and several other ROS and RNS can be generated subsequently. The candidates which pass through the mitochondrial membrane include ROS with a small number of dipoles, i.e., H2O2, HO2•, ONOOH, •OH, and •NO. The results show that the dipole moment of •NO2 is 0.35 D, indicating permeability; however, •NO2 can be eliminated quickly. The dipole moments of •OH (1.67 D) and ONOOH (1.77 D) indicate that they might be permeable. This study also suggests that the mitochondria play a central role in protecting against further oxidative stress in cells. The amounts, the long half-life, the diffusion distance, the Pm, the one-electron reduction potential, the pKa, and the rate constants for the reaction with ascorbate and glutathione are listed for various ROS/RNS, •OH, singlet oxygen (1O2), H2O2, O2•-, HO2•, •NO, •NO2, ONOO-, and ONOOH, and compared with those for H2O and oxygen (O2). Molecules with negative electrical charges cannot directly diffuse through the phospholipid bilayer of the mitochondrial membranes. Short-lived molecules, such as •OH, would be difficult to contribute to intracellular signaling. Finally, HO2• and ONOOH were selected as candidates for the ROS/RNS that pass through the mitochondrial membrane.

Oxidative stress is an essential factor for the induction of anhydrobiosis in the desiccation-tolerant midge, Polypedilum vanderplanki (Diptera, Chironomidae).

The sleeping chironomid (Polypedilum vanderplanki) is the only insect capable of surviving complete desiccation in an ametabolic state called anhydrobiosis. Here, we focused on the role of oxidative stress and we observed the production of reactive oxygen species (ROS) in desiccating larvae and in those exposed to salinity stress. Oxidative stress occurs to some extent in desiccating larvae, inducing carbonylation of proteins. Oxidative stress overcomes the antioxidant defenses of the larvae during the first hour following rehydration of anhydrobiotic larvae. It facilitates the oxidation of DNA and cell membrane lipids; however, these damages are quickly repaired after a few hours. In addition to its deleterious effects, we demonstrated that artificial exposure to oxidative stress could induce a response similar to desiccation stress, at the transcriptome and protein levels. Furthermore, the response of anhydrobiosis-related genes to desiccation and salinity stress was inhibited by antioxidant treatment. Thus, we conclude that oxidative stress is an essential trigger for inducing the expression of protective genes during the onset of anhydrobiosis in desiccating of P. vanderplanki larvae.

Evidence of Nrf2/Keap1 Signaling Regulation by Mitochodria-Generated Reactive Oxygen Species in RGK1 Cells.

It has been known that reactive oxygen species (ROS) are generated from the mitochondrial electron transport chain (ETC). Majima et al. proved that mitochondrial ROS (mtROS) caused apoptosis for the first time in 1998 (Majima et al. J Biol Chem, 1998). It is speculated that mtROS can move out of the mitochondria and initiate cellular signals in the nucleus. This paper aims to prove this phenomenon by assessing the change in the amount of manganese superoxide dismutase (MnSOD) by MnSOD transfection. Two cell lines of the same genetic background, of which generation of mtROS are different, i.e., the mtROS are more produced in RGK1, than in that of RGM1, were compared to analyze the cellular signals. The results of immunocytochemistry staining showed increase of Nrf2, Keap1, HO-1 and 2, MnSOD, GCL, GST, NQO1, GATA1, GATA3, GATA4, and GATA5 in RGK1 compared to those in RGM1. Transfection of human MnSOD in RGK1 cells showed a decrease of those signal proteins, suggesting mtROS play a role in cellular signals in nucleus.

Development of a sensitive double TaqMan Probe-based qPCR Angle-Degree method to detect mutation frequencies.

We designed a method to examine the mutation frequencies of the A3243G mutation of mitochondrial DNA (mtDNA) in patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. We performed a qPCR assay using the FAM and VIC TaqMan probes, which detect the 3243G (mutated) and 3243A (wild-type) sequences of mtDNA, respectively. The results obtained by "degree" in a series of differential mutation frequencies were used to plot a standard curve of the mutation frequency. The standard curve was then applied for qPCR assays of the desired samples. The standard deviation (%) of the samples calculated using the standard curve for the TaqMan probe was 2.4 ± 1.5%. This method could be used to examine mutation frequencies in the context of diabetes, aging, cancer, and neurodegenerative diseases.

Mitochondrial oxidative stress, mitochondrial ROS storms in long COVID pathogenesis.

Significance: This review discusses the coronavirus disease 2019 (COVID-19) pathophysiology in the context of diabetes and intracellular reactions by COVID-19, including mitochondrial oxidative stress storms, mitochondrial ROS storms, and long COVID. Recent advances: The long COVID is suffered in ~10% of the COVID-19 patients. Even the virus does not exist, the patients suffer the long COVID for even over a year, This disease could be a mitochondria dysregulation disease. Critical issues: Patients who recover from COVID-19 can develop new or persistent symptoms of multi-organ complications lasting weeks or months, called long COVID. The underlying mechanisms involved in the long COVID is still unclear. Once the symptoms of long COVID persist, they cause significant damage, leading to numerous, persistent symptoms. Future directions: A comprehensive map of the stages and pathogenetic mechanisms related to long COVID and effective drugs to treat and prevent it are required, which will aid the development of future long COVID treatments and symptom relief.

Translocation of HSP47 and generation of mitochondrial reactive oxygen species in human neuroblastoma SK-N-SH cells following electron and X-ray irradiation.

Generation of mitochondrial reactive oxygen species (ROS), lipid peroxidation, 4-hydroxy-2-nonenal, and heat-shock protein (HSP) 47 after electron and X-ray irradiations were detected in the human neuroblastoma cell line SK-N-SH. After 10 Gy electron irradiation and 15 Gy X-ray irradiation, mitochondrial ROS production and lipid peroxidation were significantly increased. Additionally, we observed a significant increase in the levels of HSP47 after 3 and 10 Gy electron irradiation as well as 15 Gy X-ray irradiation. Furthermore, myristoylation and farnesylation were increased after 10 Gy electron and 15 Gy X-ray irradiations. We found that the level of HSP47 increased in the mitochondria after 10 Gy electron and 15 Gy X-ray irradiations. HSP47 coexisted with myristoylation and farnesylation. Furthermore, HSP47 overexpression increased mitochondrial ROS production. These results suggest that HSP47 plays an important role in mitochondria and induces mitochondrial ROS production in SK-N-SH cells.

Effects of low-dose X-ray irradiation on melanin-derived radicals in mouse hair and skin.

To clarify a possible index for long-term and low-dose irradiation, the effects of repeated low-dose X-ray irradiation on the amount of melanin-derived radicals in mouse hair and tail skin were investigated. Eight-week-old female C3H/HeSlc mice were irradiated by X-rays at a dose of 100 mGy/day 5 days/week for 12 weeks. Similarly, a 4-week irradiation experiment was carried out at 500 mGy/day for C3H/HeSlc mice, or at 10, 100, and 500 mGy/day for 8-week-old female C57BL/6NCrSlc mice. The hair sample (~10 mg) was weighed accurately and stuffed into a plastic tube. The 2-cm tip of the tail was sampled and lyophilized. Melanin-derived radicals in hair and tail samples were measured by X-band electron paramagnetic resonance spectrometry. After X-ray irradiation at 100 mGy/day for 12 weeks, no difference was found in the amount of melanin-derived radicals in the hair of the irradiated and non-irradiated groups. X-ray irradiation at 500 mGy/day for 4 weeks increased the amount of melanin-derived radicals in hair compared with the non-irradiated group, but the baseline amount of melanin-derived radicals in hair was varied. The amount of melanin-derived radicals in the tail skin dose-dependently increased. Melanin-derived radicals in skin may be an endogenous marker for long-term and low-dose irradiation.

Author Correction: Changes in mitochondrial homeostasis and redox status in astronauts following long stays in space.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhancement of membrane lipid peroxidation in lung cancer cells irradiated with monoenergetic X-rays at the K-shell resonance absorption peak of phosphorus.

The aim of this study was to determine whether membrane lipid peroxidation in mammalian cells is enhanced by X-ray irradiation at the K-shell resonance absorption peak of phosphorus. A549 and wild-type p53-transfected H1299 (H1299/wtp53) cell lines derived from human lung carcinoma were irradiated with monoenergetic X-rays at 2.153 keV, the phosphorus K-shell resonance absorption peak, or those at 2.147 or 2.160 keV, which are off peaks. Immunofluorescence staining for 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, was used as marker for protein modification. In both cell lines, the HNE production was significantly enhanced after irradiation at 2.153 keV compared to sham-irradiation. The enhancement (E) was calculated as the ratio of the fluorescence intensity of irradiated cells to that of sham-irradiated cells. In both the cell lines, E2.153 was significantly larger than E2.147 and no significant difference between E2.147 and E2.160 was observed. The extra enhancement at 2.153 keV was possibly caused by energy transition within the phosphorus K-shell resonance absorption. Our results indicate that membrane lipid peroxidation in cells is enhanced by the Auger effect after irradiation at the K-shell resonance absorption peak of phosphorus rather than by the photoelectric effect of the constituent atoms in the membrane lipid at 2.147 keV.

5-Aminolevulinic acid induced apoptosis via oxidative stress in normal gastric epithelial cells.

5-Aminolevulinic acid, a precursor of heme, is utilized in a variety of applications including cancer treatment, surgery, and plant nutrition. However, 5-aminolevulinic acid itself induces oxidative stress and subsequent lipid peroxidation. Reactive oxygen species are factors in oxidative stress, not only causing cellular injury but also inducing several signal transduction pathways. Especially in cancer cells, a significant amount of signalling activation and subsequent activation of protein is caused by the enhancement of reactive oxygen species production. Reactive oxygen species levels in normal cells are low and an oxidative condition is harmful; hence, administration of 5-aminolevulinic acid to normal cells may induce oxidative stress, resulting in cell death. In this study, we investigated the effect of 5-aminolevulinic acid on normal and cancer cells with regard to oxidative stress. We used the rat normal gastric cell line RGM and its cancer-like mutant cell line RGK. 5-Aminolevulinic acid treatment of RGM cells enhanced reactive oxygen species generation and induced apoptosis associated with p53, whereas RGK cells were unaffected. In addition, RGM cell viability was recovered by application of N-acetyl-l-cysteine or p53 inhibitor. These results suggest that 5-aminolevulinic acid causes oxidative stress in normal gastric cells and induces apoptosis via the p53-dependent pathway.

Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function.

Mitochondria are a major source of intracellular energy and reactive oxygen species in cells, but are also increasingly being recognized as a controller of cell death. Here, we review evidence of signal transduction control by mitochondrial superoxide generation via the nuclear factor-κB (NF-κB) and GATA signaling pathways. We have also reviewed the effects of ROS on the activation of MMP and HIF. There is significant evidence to support the hypothesis that mitochondrial superoxide can initiate signaling pathways following transport into the cytosol. In this study, we provide evidence of TATA signal transductions by mitochondrial superoxide. Oxidative phosphorylation via the electron transfer chain, glycolysis, and generation of superoxide from mitochondria could be important factors in regulating signal transduction, cellular homeostasis, and cell death.

Changes in mitochondrial homeostasis and redox status in astronauts following long stays in space.

The effects of long-term exposure to extreme space conditions on astronauts were investigated by analyzing hair samples from ten astronauts who had spent six months on the International Space Station (ISS). Two samples were collected before, during and after their stays in the ISS; hereafter, referred to as Preflight, Inflight and Postflight, respectively. The ratios of mitochondrial (mt) to nuclear (n) DNA and mtRNA to nRNA were analyzed via quantitative PCR. The combined data of Preflight, Inflight and Postflight show a significant reduction in the mtDNA/nDNA in Inflight, and significant reductions in the mtRNA/nRNA ratios in both the Inflight and Postflight samples. The mtRNA/mtDNA ratios were relatively constant, except in the Postflight samples. Using the same samples, the expression of redox and signal transduction related genes, MnSOD, CuZnSOD, Nrf2, Keap1, GPx4 and Catalase was also examined. The results of the combined data from Preflight, Inflight and Postflight show a significant decrease in the expression of all of the redox-related genes in the samples collected Postflight, with the exception of Catalase, which show no change. This decreased expression may contribute to increased oxidative stress Inflight resulting in the mitochondrial damage that is apparent Postflight.

Reactive oxygen species induced by non-steroidal anti-inflammatory drugs enhance the effects of photodynamic therapy in gastric cancer cells.

Photodynamic therapy is useful for the treatment of cancer because it is minimally invasive for patients. Certain porphyrin compounds and their derivatives have been used as the photosensitizer because they accumulate specifically in cancerous tissues. However, the detailed mechanism of this phenomenon has not been clarified. We previously reported that a proton-coupled folate transporter, HCP1, transported porphyrins and that regulation of the protein was associated with cancer-specific reactive oxygen species from mitochondria (mitROS). Therefore, over-generation of mitROS could increase HCP1 expression and the effect of photodynamic therapy. We investigated whether pretreatment with indomethacin influenced photodynamic therapy by using a rat normal gastric mucosal cell line, RGM1, its cancer-like mutated cell line, RGK1, and a manganese superoxide dismutase (MnSOD)-overexpressing RGK cell line, RGK-MnSOD. Indomethacin promotes the generation of cellular mitROS by inhibiting the electron transport chain, and MnSOD scavenges the mitROS. We elucidated that indomethacin enhanced cancer-specific mitROS generation and increased HCP1 expression. Furthermore, RGK1 cells showed higher cellular incorporation of hematoporphyrin and better therapeutic effect with indomethacin treatment whereas RGK-MnSOD cells did not show a difference. Thus, we concluded that indomethacin improved the effect of photodynamic therapy by inducing increased mitROS generation in cancer cells.

Clinical Phenotype and Segregation of Mitochondrial 3243A>G Mutation in 2 Pairs of Monozygotic Twins.

IMPORTANCE: The regulatory factors explaining the wide spectrum of clinical phenotypes for mitochondrial 3243A>G mutation are not known. Crosstalk between nuclear genes and mitochondrial DNA might be one factor. OBSERVATIONS: In this case series, we compared 2 pairs of male twins with the mitochondrial 3243 A>G mutation and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome with a female control patient. One pair of monozygotic twins presented with diabetes and deafness in their 30s, stroke-like episodes in their 40s, and cardiac events and death in their 50s. Another pair of twins presented with deafness and stroke-like episodes in their 20s. The degree of heteroplasmy of 3243A>G mutation in the various tissues and organs was similar in the first pair of twins compared with the control patient. CONCLUSIONS AND RELEVANCE: The clinical phenotype and segregation of mitochondrial 3243A>G mutation was similar in monozygotic twins. The onset age and distribution of the symptoms might be regulated by nuclear genes. Our findings might help to predict the clinical course of the surviving twins and afford an opportunity for therapy before the onset of mitochondrial disease, especially for monozygotic twins caused by nuclear transfer with a small amount of nuclear-donor mitochondrial DNA.

Chasing great paths of Helmut Sies "Oxidative Stress".

Prof. Dr. Helmut Sies is a pioneer of "Oxidative Stress", and has published over 18 papers with the name of "Oxidative Stress" in the title. He has been Editor-in-Chief of the journal "Archives of Biochemistry and Biophysics" for many years, and is a former Editor-in-Chief of the journal "Free Radical Research". He has clarified our understanding of the causes of chronic developing diseases, and has studied antioxidant factors. In this article, importance of "Oxidative Stress" and our mitochondrial oxidative stress studies; roles of mitochondrial ROS, effects of vitamin E and its homologues in oxidative stress-related diseases, effects of antioxidants in vivo and in vitro, and a mitochondrial superoxide theory for oxidative stress diseases and aging are introduced, and some of our interactions with Helmut are described, congratulating and appreciating his great path.

MnSOD downregulation induced by extremely low 0.1 mGy single and fractionated X-rays and microgravity treatment in human neuroblastoma cell line, NB-1.

A human neuroblastoma cell line, NB-1, was treated with 24 h of microgravity simulation by clinostat, or irradiated with extremely small X-ray doses of 0.1 or 1.0 mGy using single and 10 times fractionation regimes with 1 and 2 h time-intervals. A quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) examination was performed for apoptosis related factors (BAX, CYTC, APAF1, VDAC1-3, CASP3, CASP8, CASP9 P53, AIF, ANT1 and 2, BCL2, MnSOD, autophagy related BECN and necrosis related CYP-40. The qRT-PCR results revealed that microgravity did not result in significant changes except for a upregulation of proapoptotic VDAC2, and downregulations of proapoptotic CASP9 and antiapoptotic MnSOD. After 0.1 mGy fractionation irradiation, there was increased expression of proapoptotic APAF1 and downregulation of proapoptotic CYTC, VDAC2, VDAC3, CASP8, AIF, ANT1, and ANT2, as well as an increase in expression of antiapoptotic BCL2. There was also a decrease in MnSOD expression with 0.1 mGy fractionation irradiation. These results suggest that microgravity and low-dose radiation may decrease apoptosis but may potentially increase oxidative stress.

Manganese superoxide dismutase promotes interaction of actin, S100A4 and Talin, and enhances rat gastric tumor cell invasion.

It has been demonstrated that cancer cells are under high levels of oxidative stress and express high levels of Manganese superoxide dismutase (MnSOD) to protect themselves and support the anabolic metabolism needed for growth and cell motility. The aim of this study was to identify proteins that may have a correlation with invasion and redox regulation by mitochondrial reactive oxygen species (ROS). MnSOD scavenges superoxide anions generated from mitochondria and is an important regulator of cellular redox status. Oxidative posttranslational modification of cysteine residues is a key mechanism that regulates protein structure and function. We hypothesized that MnSOD regulates intracellular reduced thiol status and promotes cancer invasion. A proteomic thiol-labeling approach with 5-iodoacetamidofluorescein was used to identify changes in intracellular reduced thiol-containing proteins. Our results demonstrate that overexpression of MnSOD maintained the major structural protein, actin, in a reduced state, and enhanced the invasion ability in gastric mucosal cancer cells, RGK1. We also found that the expression of Talin and S100A4 were increased in MnSOD-overexpressed RGK1 cells. Moreover, Talin bound not only with actin but also with S100A4, suggesting that the interaction of these proteins may, in part, contribute to the invasive ability of rat gastric cancer.

A mitochondrial superoxide theory for oxidative stress diseases and aging.

Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed "the Superoxide Theory," which postulates that superoxide (O2 (•-)) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q10) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich's seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

Mitochondrial reactive oxygen species accelerate the expression of heme carrier protein 1 and enhance photodynamic cancer therapy effect.

Photodynamic therapy using hematoporphyrin and its derivatives is clinically useful for cancer treatments. It has been reported that cancer cells incorporate hematoporphyrin and its derivatives via heme carrier protein 1, which is a proton-coupled folate transporter. However, the mechanism of this protein expression has not been elucidated. In general, the concentration of reactive oxygen species in cancer cells is higher than that in normal cells. We previously reported that reactive oxygen species from mitochondria involved in the expression of peptide transporter 1 and accelerate the uptake of 5-aminolevulinic acid, which is a precursor of protoporphyrin IX. We suggested mitochondrial reactive oxygen species also regulated the expression of heme carrier protein 1. In this study, we used a rat gastric mucosal cell line RGM1 and its cancer-like mutated cell line RGK1. We clarified the expression of heme carrier protein 1 increased in cancer cells and it decreased in manganese superoxide dismutase expressed cancer cells. In addition, the uptake level of hematoporphyrin and photodynamic therapeutic effect were also decreased in manganese superoxide dismutase expressed cancer cells in comparison with cancer cells. Thus, we concluded that mitochondrial reactive oxygen species regulated heme carrier protein 1 expression and photodynamic therapeutic effect.