Obacunone protects retinal pigment epithelium cells from ultra-violet radiation-induced oxidative injury.

Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.

Cell inactivation and double-strand breaks: the role of core ionizations, as probed by ultrasoft X rays.

The large RBE (approximately 7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649-658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.

Single- and double-strand breaks in pBR322 plasmid DNA by monochromatic X-rays on and off the K-absorption peak of phosphorus.

Using a synchrotron irradiation system pBR322 plasmid DNA was irradiated under vacuum by monochromatic X-rays having five specific photon energies (2.147, 2.153, 2.159, 2.168 and 2.199 keV) both on and off the K-absorption peak (2.153 keV) of phosphorus. The single- and double-strand breaks (ssb and dsb) were measured as conversions of the closed circular form of DNA (form I) to open circular (form II) and linear (form III) forms respectively. Exposures to induce one strand break per molecule were lowest at the peak (2.153 keV), and highest at 2.147 keV; the ratios were 2.7 for ssb and 3.0 for dsb. The exposures for dsb were 21-26 times higher than those for ssb. When the exposures were converted to absorbed doses in grays the absorbed doses per ssb were almost independent of photon energy. This result indicates that a certain absorbed dose was necessary to induce a ssb, regardless of whether photons were absorbed by the K-shell of phosphorus or by other shells, or by other atoms. However, the absorbed dose per dsb at 2.147 keV was 1.17 times higher than that averaged over four X-ray energies above 2-153 keV, indicating that the K-shell absorption, and the subsequent Auger event, efficiently induce dsb. The results are also discussed concerning the number of photo-absorptions of the constituent atoms per DNA strand break.

Asymmetric fusion between protoplasts of tomato (Lycopersicon esculentum Mill.) and gamma-irradiated protoplasts of potato (Solanum tuberosum L.): the effects of gamma irradiation.

This paper describes the aggregation of nuclei in heterokaryons of tomato and unirradiated or irradiated potato protoplasts and the effects of gamma irradiation of potato and tomato protoplasts on single- and double-stranded DNA fragmentation, DNA repair and DNA synthesis as revealed by alkaline and pulsed field gel electrophoresis and an immunocytochemical technique. The prospects for obtaining highly asymmetric somatic hybrids of tomato and gamma-irradiated potato are discussed.

Single-strand breaks in the DNA of human cells exposed to visible light from phototherapy lamps in the presence and absence of bilirubin.

Clinical evidence indicates that phototherapy of hyperbilirubinaemia in newborn infants is a safe and efficient form of therapy. The short-term side effects are not serious and seem to be well controlled. There are few long-term follow-up studies of phototherapy-treated infants. Therefore one cannot completely exclude the possibility that side effects can be found in future studies. With this background we undertook the present study of possible genotoxic effects of phototherapy. Human cells of the established glioblastoma cell line TMG-1 were used. The cells were exposed to visible light in the presence of different concentrations of bilirubin or in the absence of bilirubin. DNA was unwound in alkaline solution and the induction of strand breaks was assayed by a method taking advantage of the fluorescence from the dye Hoechst 33258. Blue light induced single-strand breaks in the DNA of cells in culture in the absence of bilirubin. During irradiation of bilirubin solutions with blue and green phototherapy light, long-lived toxic photoproducts were formed under in vitro conditions. At high and clinically relevant bilirubin concentrations, the effects of blue and green light were relatively similar. At low concentrations, there was a smaller effect of green light as expected from the absorption spectrum of bilirubin. It remains to be seen whether the genotoxic effect observed in the present studies can occur in vivo.

Combined electron radiation and hyperthermia. Repair of DNA strand breaks in NHIK 3025 cells irradiated and incubated at 37, 42.5, or 45 degrees C.

Induction of DNA strand breaks by a short electron pulse (18.5 Gy) and the subsequent strand-break rejoining were investigated at hyperthermia (42.5 and 45 degrees C) and at 37 degrees C during irradiation and repair. The cells were irradiated immediately after 2.5 min equilibration (i.e., from 37 to 42.5 or 45 degrees C) to investigate the effect of short-duration hyperthermia on radiation damage and subsequent repair. Due to a high radiation dose rate and a rapid lysis technique, the cells could be kept at the actual temperature during irradiation and repair, and the strand-break frequency could be measured only seconds after irradiation. At all temperatures, a constant or possible increase in the initial number of breaks was observed during the first 7 sec after the electron pulse. At 37 degrees C, strand-break rejoining was nearly complete within 1 hr. Hyperthermia at 42.5 degrees C had only minor influence on the net rate of strand-break rejoining. At 45 degrees C, 50% of the breaks remained after 1 hr. Subsequent incubation for 23 hr at 37 degrees C reduced by half the number of breaks remaining at 1 hr in irradiated samples. Unirradiated samples exposed to the same heat treatment showed a significant increase in the number of DNA strand breaks. Thus, heat treatment at 45 degrees C may lead to a combined effect of reduced rejoining capacity and formation of breaks after the electron pulse which in turn may be responsible for increased cell death when both modalities are employed.

A spectrum response study on single strand DNA breaks, sister chromatid exchanges, and lethality induced by phototherapy lights.

Little information is available on the effect of visible light from commercial fluorescence lamps, commonly used in the treatment of neonatal hyperbilirubinemia, on parameters related to genetic damage in eucaryotic cells. The present study was undertaken to determine whether or not visible light of different wavelengths had any differential effects on the frequency of DNA breaks (frank breaks plus alkaline labile lesions), DNA replication, frequency of sister chromatid exchanges, and survival in cultured Chinese hamster cells. The results revealed that the "blue" spectral band (420 to 500 nm) is mainly responsible for DNA breaks, sister chromatid exchanges, and lethality induced by fluorescent light. This band is precisely that which bilirubin heavily absorbs and, hence, is the most efficient for the decomposition of this metabolite. These results were obtained with the use of light doses of 5 to 30 X 10(4) J/m3 versus the light doses of the order of 100 X 10(4) J/m2 being received by infants undergoing phototherapy treatments.

[Action of carminomycin on the structural integrity, primary damage susceptibility and postradiation regeneration of the DNA from Erhlich's ascitic carcinoma]. / Deistvie karminomitsina na strukturnuiu tselostnost', pervichnuiu radiatsionnuiu porazhaemost' i postradiatsionnuiu reparatsiiu DNK astsitnoi kartsinomy Erlikha.

Carminomycin added to the cell culture of Ehrlichs ascites carcinoma (EAC) induced single breaks in DNA whose number increased proportionally to the amount of the antibiotic and the incubation time with it (up to 1 hour). The relationship between the incubation time and DNA injury is first of all defined by the necessity for the antibiotic metabolic activation or the properties of endonucleases. No increase in the number of one-thread breaks during the incubation period of 60 to 120 minutes indicated a possible reparation of DNA injury induced by the antibiotic in the late periods. The exposure of the EAC cells to carminomycin before gamma-irradiation did not result in any increase in the primary radiation injury to DNA but suppressed DNA postradiation reparation.

[Effect of a DNA-rubomycin complex on the molecular mass, radiosensitivity and reparation of gamma-induced single-stranded breaks in the DNA of sarcoma 180 cells]. / Vliianie kompleksa DNK-rubomitsin na molekuliarnuiu massu, radiochuvstvitel'nost'i reparatsiiu gamma-indutsirovannykh odnonitevykh razryvov v DNK kletok sarkomy 180.

The use of the DNA-rubomycin complex resulted in appearance of one-thread breaks (OB) in DNA and markedly increased the number of the DNA OB determined immediately after irradiation. The DNA-rubomycin complex decreased the volume of the repaired OB and induced postreparation degradation of the one-bond DNA (oDNA) in the sarcoma 180 cells. The effect of the decrease in the molecular mass of the oDNA 6 hours after the irradiation to the level of the initial damage is probably in favour of the fact that degradation takes place in the areas of the repaired breaks. The data indicated that the viscosimetric method for the analysis of the OB in DNA may be promising in estimation of the efficacy of radiation and chemotherapy of solid tumors.

[Mechanism of the mutagenic action of the decay of incorporated phosphorus-32]. / O mikhanizme mutagennogo deistviia raspada inkorporirovannogo fosfora-32.

The experimental material concerning that physico-chemical consequences of 32P decay in the molecular structure of DNA, their reparation mechanism as well, and resulting mutagenic effects have been analysed. The reparation of the single-strand break of the DNA chain does not cause the changes of microdeletion and microinsertion type but instead of the changes observed are of the nucleic bases conversion type. It is concluded that the mutations caused by the decay of 32P incorporated appear as a result of errors in the selection of nucleic bases during the reparative replication of the non-conservative type.