Mitochondrial Effects in the Liver of C57BL/6 Mice by Low Dose, High Energy, High Charge Irradiation.

Galactic cosmic rays are primarily composed of protons (85%), helium (14%), and high charge/high energy ions (HZEs) such as 56Fe, 28Si, and 16O. HZE exposure is a major risk factor for astronauts during deep-space travel due to the possibility of HZE-induced cancer. A systems biology integrated omics approach encompassing transcriptomics, proteomics, lipidomics, and functional biochemical assays was used to identify microenvironmental changes induced by HZE exposure. C57BL/6 mice were placed into six treatment groups and received the following irradiation treatments: 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), 350 MeV/n 28Si (0.2 Gy), 137Cs (1.0 Gy) gamma rays, 137Cs (3.0 Gy) gamma rays, and sham irradiation. Left liver lobes were collected at 30, 60, 120, 270, and 360 days post-irradiation. Analysis of transcriptomic and proteomic data utilizing ingenuity pathway analysis identified multiple pathways involved in mitochondrial function that were altered after HZE irradiation. Lipids also exhibited changes that were linked to mitochondrial function. Molecular assays for mitochondrial Complex I activity showed significant decreases in activity after HZE exposure. HZE-induced mitochondrial dysfunction suggests an increased risk for deep space travel. Microenvironmental and pathway analysis as performed in this research identified possible targets for countermeasures to mitigate risk.

C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines.

A small nuclear protein, C1D, has roles in various cellular processes, transcription regulation, genome stability surveillance, DNA repair and RNA processing, all of which are required to maintain the host life cycles. In the previous report, C1D directly interacts with XPB, a component of the nucleotide excision repair complex, and C1D knockdown reduced cell survival of 27-1 cells, CHO derivative cells, after UV irradiation. To find out the role of C1D in UV-damaged cells, we used human cell lines with siRNA or shRNA to knockdown C1D. C1D knockdown reduced cell survival rates of LU99 and 786-O after UV irradiation, although C1D knockdown did not affect the efficiency of the nucleotide excision repair. Immunostaining data support that C1D is not directly involved in the DNA repair process in UV-damaged cells. However, H2O2 treatment reduced cell viability in LU99 and 786-O cells. We also found that C1D knockdown upregulated DDIT3 expression in LU99 cells and downregulated APEX1 in 786-O cells, suggesting that C1D functions as a co-repressor/activator. The data accounts for the reduction of cell survival rates upon UV irradiation.

[RADIOPROTECTIVE ACTIVITY OF POLYMETOXY-LATED FLAVONOIDS OF CITRUS EXTRACT].

The aim of the study was to establish the radioprotective activity of citrus polymetoxylated flavonoids extract (CPMFE) on the X-irradiated rats. The experiments were carried out on white Wistar rats. Animals were irradiated with X rays in doses of 5 Gy and 7 Gy. The control group consisted the sham-irradiated rats. Part of animals of each group were treated with intramusculary injections of CPMFE (dose 30 mg/kg) during 7 days; blood was taken from the tail vein (0.5 ml) for detection of lipoperoxides (LOO.) content. On the 3rd day after irradiation 3 animals from each group were sacrificed (under ether anesthesia) and blood samples were taken for the study of antioxidant status. The activity of antioxidant enzymes (catalase (CAT) and superoxidedismutase (SOD)) was determined by the spectrophotometric method; the content of LOO.in the blood was determined by electron paramagnetic resonance (EPR) mrthod. In group of irradiated rats a sharp dose-dependent inactivation of blood antioxidant enzymes (SOD, CAT) and intensification of the lipid peroxidation were detected. The direct and feedback mechanism in the regulation of CAT and SOD activity, ensuring the implementation of antioxidant protection in the body was revealed. Under irradiation with 7Gy rapid death of animals (on 3-d day after irradiation the mortality of animals was 70%, and on the 5th day all died) were detected. During irradiation with dose 5 Gy the survival of animals increased (on the 8-th day after irradiation - 50% survival rate). CPMFE in dose-dependent manner supported the reduce the intensity of lipid peroxidation processes - at relatively low doses of radiation (5Gy) during the first 3 days the content of LOO.in the blood decreased insignificantly compared with indices in untreated animals, whereas with an increase in the dose of irradiation (7Gy) a statistically significant antiradical effect of CPMFE (a statistically significant decrease in the LOO. content) was detected. Under the effect of CPMFE in the blood of rats irradiated with a dose of 5 Gy and 7 Gy, the activity of CAT and SOD, not statistically significant tends to increase (more significant with a dose of 7 Gy). CPMFE did not affect the cumulative survival of animals irradiated with a dose of 5 Gy, but reduced the mortality of rats by 20% (on the 3rd day of irradiation), and contributed to an increase in the life expectancy of animals by 2 times (up to 7 days) in the case of dose 7 Gr. Based on the analysis of the research results, it can be assumed that under conditions of radiation damage, exogenous antioxidants synergistically with a dose-dependently activated endogenous non-enzymatic antioxidant system of the body (especially at 7Gy) contribute to the effective suppression of chain reactions of peroxidation, reduction of mortality and increase in life expectancy of animals.

GUCY2C Signaling Opposes the Acute Radiation-Induced GI Syndrome.

High doses of ionizing radiation induce acute damage to epithelial cells of the gastrointestinal (GI) tract, mediating toxicities restricting the therapeutic efficacy of radiation in cancer and morbidity and mortality in nuclear disasters. No approved prophylaxis or therapy exists for these toxicities, in part reflecting an incomplete understanding of mechanisms contributing to the acute radiation-induced GI syndrome (RIGS). Guanylate cyclase C (GUCY2C) and its hormones guanylin and uroguanylin have recently emerged as one paracrine axis defending intestinal mucosal integrity against mutational, chemical, and inflammatory injury. Here, we reveal a role for the GUCY2C paracrine axis in compensatory mechanisms opposing RIGS. Eliminating GUCY2C signaling exacerbated RIGS, amplifying radiation-induced mortality, weight loss, mucosal bleeding, debilitation, and intestinal dysfunction. Durable expression of GUCY2C, guanylin, and uroguanylin mRNA and protein by intestinal epithelial cells was preserved following lethal irradiation inducing RIGS. Oral delivery of the heat-stable enterotoxin (ST), an exogenous GUCY2C ligand, opposed RIGS, a process requiring p53 activation mediated by dissociation from MDM2. In turn, p53 activation prevented cell death by selectively limiting mitotic catastrophe, but not apoptosis. These studies reveal a role for the GUCY2C paracrine hormone axis as a novel compensatory mechanism opposing RIGS, and they highlight the potential of oral GUCY2C agonists (Linzess; Trulance) to prevent and treat RIGS in cancer therapy and nuclear disasters. Cancer Res; 77(18); 5095-106. ©2017 AACR.

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation.

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production.

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

Multiple parametric approaches to assess acute radiation lung injury of rats radiation lung injury of rats.

The effect of whole body gamma irradiation (WBI) in single fraction was studied, as well as its influence on the secretion of various biochemical markers and cellular component that could be used as acute radiation lung injury marker. Sprague dawley rats were treated with WBI (60Co) of radiation dose from 1 Gy to 5 Gy (dose rate - 0.95 Gy/min). Bronchoalveolar lavage fluid was retrieved from all animals in control and radiation treated groups up to 72 h post radiation. Bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH ), acid phosphatase (AP ), alkaline phosphatase (ALP ), cell count and total protein. Intragroup and intergroup comparison of BALF parameters at different radiation doses showed significant difference. LDH was significantly increased as the dose increased from 1Gy to 5Gy (P = 0.00) after 2 h with effect size of difference (r > 0.3). ALP was significantly altered after 3Gy and 4Gy (P < 0.05). AP was significantly altered at 2Gy-5Gy (p < 0.05). Total protein level changed significantly from 1Gy to 5Gy (P < 0.00). Cellular content of BALF showed significant changes after radiation exposure. BALF parameters like LDH, AP, ALP, neutrophils, lymphocytes, total leukocyte count and total protein were sensitive to radiation exposure and their levels vary significantly up to 72 h after single whole body radiation exposure in Sprague dawley rats. It can be concluded that the biochemical indices in BALF have more wide application in evaluation of acute radiation induced lung injury.

Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17.

Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17.

Ciprofloxacin Therapy Results in Mitigation of ATP Loss after Irradiation Combined with Wound Trauma: Preservation of Pyruvate Dehydrogenase and Inhibition of Pyruvate Dehydrogenase Kinase 1.

Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited with ciprofloxacin treatment, thus enabling PDH to increase ATP production. Increased ileum levels of pyruvate dehydrogenase kinase 1 protein (PDK1, an enzyme responsible for PDH phosphorylation) after combined injury were also prevented by ciprofloxacin treatment. Taken together, these data suggest that ciprofloxacin oral administration after combined injury had a role in sustained ileum ATP levels, and may have acted through preservation of PDH by HSP-70 and inhibition of PDK1. These molecular changes in the ileum are simply one of a host of mechanisms working in concert with one another by which ciprofloxacin treatment mitigates body weight loss and drastically enhances subsequent survival after combined injury. To this end, our findings indicate that oral treatment of ciprofloxacin is a valuable therapeutic treatment after irradiation with combined injury and warrants further analyses to elucidate the precise mechanisms involved.

SKI2162, an inhibitor of the TGF-ß type I receptor (ALK5), inhibits radiation-induced fibrosis in mice.

Here we demonstrated that SKI2162, a small-molecule inhibitor of the TGF-ß type I receptor (ALK5), prevented radiation-induced fibrosis (RIF) in mice. SKI2162 inhibited phosphorylation of Smad and induction of RIF-related genes in vitro. In RIF a mouse model, SKI2162 reduced late skin reactions and leg-contracture without jeopardizing the acute skin reaction. Irradiation of mouse tissue increased COL1A2 mRNA levels, and topical administration of SKI2162 significantly inhibited this effect. Thus, these findings support that SKI2162 has potential value as novel RIF-protective agent, and could be candidate for clinical trials.

Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

Radiation-induced damage to the retina triggers leukostasis, retinal endothelial cell (REC) death, and subsequent hypoxia. Resultant ischemia leads to visual loss and compensatory retinal neovascularization (RNV). Using human RECs, we demonstrated that radiation induced leukocyte adhesion through mechanisms involving p38MAPK, p53, and ICAM-1 activation. Additional phenotypic changes included p38MAPK-dependent tyrosine phosphorylation of the focal adhesion scaffolding protein, paxillin (Tyr118). The quinic acid derivative KZ-41 lessened leukocyte adhesion and paxillin-dependent proliferation via inhibition of p38MAPK-p53-ICAM-1 signaling. Using the murine oxygen-induced retinopathy (OIR) model, we examined the effect of KZ-41 on pathologic RNV. Daily ocular application of a KZ-41-loaded nanoemulsion significantly reduced both the avascular and neovascular areas in harvested retinal flat mounts when compared to the contralateral eye receiving vehicle alone. Our data highlight the potential benefit of KZ-41 in reducing both the retinal ischemia and neovascularization provoked by genotoxic insults. Further research into how quinic acid derivatives target and mitigate inflammation is needed to fully appreciate their therapeutic potential for the treatment of inflammatory retinal vasculopathies.

Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

Time evolution of active caspase-3 labelling after in vivo exposure to UVR-300 nm.

PURPOSE: To determine the time evolution of active caspase-3 protein expression in albino rat lens after in vivo exposure to low-dose UVR-300 nm, as detected by immunofluorescence. METHODS: Forty Sprague-Dawley rats were unilaterally exposed in vivo to 1 kJ/m(2) UVR-300 nm for 15 min. At 0.5, 8, 16 and 24 hr after the UVR exposure, the exposed and contralateral nonexposed lenses were removed and processed for immunohistochemistry. Three mid-sagittal sections from each lens were stained. The cells labelled for active caspase-3 in each section of both the exposed and nonexposed lenses were counted and recorded three times. The difference of the proportion of labelling between the exposed and contralateral nonexposed lenses within each animal was calculated. The differences of active caspase-3 labelling at four different time-points after exposure were used to determine the time evolution of active caspase-3 expression. RESULTS: Caspase-3 expression was higher in the exposed than in contralateral nonexposed lenses. The mean difference between the exposed and contralateral nonexposed lenses, including all lenses from all time intervals, was 0.12 ± 0.01 (= CI 95%). The mean differences between the exposed and contralateral nonexposed lenses were 0.11 ± 0.02, 0.13 ± 0.02, 0.14 ± 0.01 and 0.09 ± 0.03 (= CI 95%) for the 0.5-, 8-, 16- and 24-hr time groups, respectively. The orthogonal comparison showed no difference in the expression of active caspase-3 between the 0.5- and the 24-hr groups (Test statistic 1.50, F1,36 = 4.11, p < 0.05) or between the 8- and the 16-hr groups (test statistic 0.05, F1,36 = 4.11, p < 0.05). There was a difference when comparing the 0.5- and 24-hr groups to the 8- and 16-hr groups (test statistic 7.01, F1,36 = 4.11, p < 0.05). CONCLUSION: The expression of active caspase-3 in the lens epithelium increases after UVR exposure. There is a peak of expression approximately 16 hr after the exposure.

Low-dose radiation modifies skin response to acute gamma-rays and protons.

The goal of the present study was to obtain pilot data on the effects of protracted low-dose/low-dose-rate (LDR) γ-rays on the skin, both with and without acute gamma or proton irradiation (IR). Six groups of C57BL/6 mice were examined: a) 0 Gy control, b) LDR, c) Gamma, d) LDR+Gamma, e) Proton, and f) LDR+Proton. LDR radiation was delivered to a total dose of 0.01 Gy (0.03 cGy/h), whereas the Gamma and Proton groups received 2 Gy (0.9 Gy/min and 1.0 Gy/min, respectively). Assays were performed 56 days after exposure. Skin samples from all irradiated groups had activated caspase-3, indicative of apoptosis. The significant (p<0.05) increases in immunoreactivity in the Gamma and Proton groups were not present when LDR pre-exposure was included. However, the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay for DNA fragmentation and histological examination of hematoxylin and eosin-stained sections revealed no significant differences among groups, regardless of radiation regimen. The data demonstrate that caspase-3 activation initially triggered by both forms of acute radiation was greatly elevated in the skin nearly two months after whole-body exposure. In addition, LDR γ-ray priming ameliorated this response.

Semiquinone derivative isolated from Bacillus sp. INM-1 protects cellular antioxidant enzymes from γ-radiation-induced renal toxicity.

This study was focused to evaluate protection of indigenous antioxidant system of mice against gamma radiation-induced oxidative stress using a semiquinone (SQGD)-rich fraction isolated from Bacillus sp. INM-1. Male C57bl/6 mice were administered SQGD (50 mg/kgb.w.i.p.) 2 h before irradiation (10 Gy) and modulation in antioxidant enzymes activities was estimated at different time intervals and compared with irradiated mice which were not pretreated by SQGD. Compared to untreated controls, SQGD pretreatment significantly (p < 0.05) accelerates superoxide dismutase, catalase, GSH, and glutathione-S-transferase activities. Similarly, significant (p < 0.05) increase in the expression of superoxide dismutase, catalase, GSH, and glutathione-S-transferase was observed in irradiated mice pretreated by SQGD, compared to only irradiated groups. Total antioxidant status equivalent to trolox was estimated in renal tissue of the mice after SQGD administration. Significant ABTS(+) radical formation was observed in H2O2-treated kidney homogenate, due to oxidative stress in the tissue. However, significant decrease in the levels of ABTS(+) radical was observed in kidney homogenate of the mice pretreated with SQGD. Therefore, it can be concluded that SQGD neutralizes oxidative stress by induction of antioxidant enzymes activities and thus improved total antioxidant status in cellular system and hence contributes to radioprotection.

Infrared irradiation alters the expression of matrix metalloproteinases and glycosaminoglycans in the cornea and crystalline lens.

BACKGROUND: Prolonged exposure to infrared (IR) radiation is associated with different types of damage to cornea and lens. The aim of our study was to investigate the effect of acute and chronic exposure to IR radiation on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 and on the expression of glycosaminoglycans (GAG) in the rabbit cornea and crystalline lens. METHODS: New Zealand rabbits were subjected to IR radiation for 4 months (chronic exposure to IR) or to normal light (control group). In experiments regarding acute exposure, animals were subjected to IR radiation or normal light for 12 h, in the presence of 0.1% diclofenac sodium (eye drops instilled in the right eye of animals) or saline (instilled in the left eye of animals). The cornea and lens were dissected away and homogenized. The activity of MMP-2 and MMP-9 was assayed by gelatine zymography. Total GAG were isolated from tissue specimens after lipid extraction and extensive digestion with pronase and DNase and characterized by treatment with GAG-degrading enzymes, followed by electrophoresis on cellulose acetate membranes. RESULTS: Acute or chronic exposure to IR radiation induced the activity of MMP-2 in cornea and lens, whereas only acute IR radiation increased the content of heparan sulphate in crystalline lens. Local administration of diclofenac sodium did not prevent the above effects of acute IR radiation. CONCLUSIONS: The detrimental effects of excessive or prolonged exposure of the eyes to IR radiation are associated with induced activity of MMP-2 in cornea and lens and alterations in the content of heparan sulphate in lens. Thus, MMP and GAG may offer alternative targets for pharmacological intervention to confront ocular damages associated with IR radiation.

A Class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo.

It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm(2) on the cornea) for 1 h using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 h. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear λ-crystallin after the in vivo UVA exposure. It is concluded that UVA-induced loss of free NADH (which fluoresces blue) may have allowed the natural yellow fluorescence of λ-crystallin and other proteins in the lens nucleus to become visible. Increased fluorescence exhibited by UVA-exposed λ-crystallin may have been the result of a UVA-induced change in the conformation of the protein occurring during the initial UVA-exposure in vivo. The results demonstrate the greater susceptibility of the lens nucleus to UVA-induced stress, and may relate to the formation of human nuclear cataract. The senofilcon A contact lens was shown to be beneficial in protecting the rabbit lens against effects of UVA light, including changes in fluorescence, increased yellowing and loss of pyridine nucleotides.

Retinol dehydrogenase 13 protects the mouse retina from acute light damage.

PURPOSE: To investigate whether retinol dehydrogenase 13 (RDH13) can protect the retina from acute light-induced damage. METHODS: We generated Rdh13 knockout mice using molecular biologic methods and assessed the associated morphological and functional changes under room-light conditions by hematoxylin-eosin (H&E), transmission electron microscopy (TEM), and scotopic electroretinography. Then, the light-damage model was established by exposure to diffuse white light (3,000 lx) for 48 h. Twenty-four h after light exposure, H&E was used for the histological evaluation. The thickness of the outer-plus-inner-segment and the outer nuclear layer was measured on sections parallel to the vertical meridian of the eye. An electroretinography test was performed to assess the functional change. Furthermore, the impairment of mitochondria was detected by TEM. Finally, the expression of cytochrome c (CytC) and other apoptosis-related proteins was detected by western blot. RESULTS: We found that there was no obvious difference in phenotype or function between Rdh13 knockout and wild-type mice. In Rdh13(-/-) mice subjected to intense light exposure, the photoreceptor outer-plus-inner-segment and outer nuclear layer were dramatically shorter, and the amplitudes of a- and b-waves under scotopic conditions were significantly attenuated. Distinctly swollen mitochondria with disrupted cristae were observed in the photoreceptor inner segments of Rdh13(-/-) mice. Increased expression levels of CytC, CytC-responsive apoptosis proteinase activating factor-1 (Apaf-1) and caspases 3, and other mitochondria apoptosis-related genes (nuclear factor-kappa B P65 [P65] and B-cell lymphoma 2-associated X protein [Bax]) were observed in Rdh13(-/-) mice. CONCLUSIONS: Rdh13 can protect the retina against acute light-induced retinopathy. The mechanism may involve inhibition of the mitochondrial apoptosis pathway.

Radiation-induced c-Jun activation depends on MEK1-ERK1/2 signaling pathway in microglial cells.

Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1ß, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation.

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression.

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-ß1 (TGF-ß)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-ß signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.