Bystander signal production and response are independent processes which are cell line dependent.

PURPOSE: Radiation-induced bystander effects are now an established phenomenon seen in numerous models; however it is not known whether the magnitude of the bystander effect is determined by the signal produced by the irradiated cells or the response of the exposed cells. The aim of this investigation is to determine the importance of the bystander signal versus the bystander response in three different cell line models. METHODS: A matrix design experiment using cell lines, HPV-G, CHO-K1 and E89 (glucose 6-phosphate dehydrogenase (G6DP) null) was set up to produce irradiated cell conditioned medium (ICCM), which was used to treat all cell lines. RESULTS: For HPV-G and CHO-K1 lines, the response to autologous ICCM was significantly different to that when treated with ICCM generated from another line. These lines displayed no response to E89 ICCM, nor did E89 cells show a significant response to any ICCM, suggesting that G6DP plays a key role in the bystander effect. CONCLUSION: These data suggest, for these cell lines at least, that in the case of cell lines capable of responding to the bystander signal, it is the signal produced by the irradiated cell that determines the magnitude of the bystander effect.

Effect of alpha-tocopherol on pro-oxidant and antioxidant enzyme status in radiation-treated oral squamous cell carcinoma.

OBJECTIVES: The relationships between alpha-tocopherol, pro-oxidant and antioxidant enzyme status, and radiation toxicity were studied in stage II, III, and IVA oral squamous cell carcinoma patients. The low levels of malondialdehyde and increased activities of antioxidant enzymes were correlated with decreased oxidative stress by alpha-tocopherol in oral cancer patients treated with radiotherapy. The objective of the present study was to evaluate the effect of alpha-tocopherol on oxidant-antioxidant enzyme status in oral squamous cell carcinoma patients treated with radiotherapy. MATERIALS AND METHODS: The study included three groups with histologically confirmed oral squamous cell carcinoma patients (untreated), and they were further divided into two groups, viz., one consisting of patients who underwent radiotherapy alone (radiotherapy was given at the dosage of 6000 cGy in five fractions per week for a period of 6 weeks); and the other group treated with radiotherapy plus alpha-tocopherol supplementation (alpha-tocopherol was supplemented at a dosage of 400 IU/day) for the entire period of radiotherapy. RESULTS: A significant decrease ( P < 0.001) in malondialdehyde levels and increase in activities of antioxidant enzymes ( P < 0.001) in hemolysate were noticed in patients treated with radiotherapy and simultaneously supplemented with alpha-tocopherol when compared to radiation-treated patients. CONCLUSION: It was seen that alpha-tocopherol played a role in protecting against the damage caused by irradiation in oral squamous cell carcinoma patients treated with radiotherapy, by enhancing the antioxidant enzyme status and reducing the pro-oxidant status.

Photosensitizing activity of advanced glycation endproducts on tryptophan, glucose 6-phosphate dehydrogenase, human serum albumin and ascorbic acid evaluated at low oxygen pressure.

A comparative study of the photosensitizing activity of advanced glycation endproducts (AGEs) prepared by incubating glucose (Glc), threose (Threo) and ascorbate (AH-) in the presence of lysine (Lys) was performed. Photochemical activity was evaluated under low oxygen pressure with the aim to simulate the conditions of the eye lens. AGE-sensitized tryptophan and AH- photodecomposition and glucose 6-phosphate dehydrogenase inactivation were studied. In all systems, glucose-derived AGEs showed the highest photosensitizing efficiency, followed by ascorbate and threose. The presence of different sensitizers in glycation products mixtures was investigated. For this purpose, Trp decomposition quantum yields were determined at 344 and 367 nm. The values obtained at 344 nm are between three and six times higher than those observed at 367 nm, confirming the presence of at least two compounds with different photosensitizing activities in the mixtures. The chemiluminescence associated with the AGE-mediated oxidation of free Trp and Trp residues in human serum albumin was also studied, and a good correlation between the emission of light and the extent of Trp decomposition was found. In conclusion, it is demonstrated that glucose derived AGEs, which can be formed in vivo in the eye lens of diabetic patients and are accumulated in elderly lenses, have a higher photosensitizing efficiency, at low oxygen pressure, than those arising from ascorbate and threose. This high efficiency is especially significant when proteins are employed as photochemical targets, indicating that protein-sensitizer interaction and the local environment around the sensitizers play an important role.

Protective role of superoxide dismutases against ionizing radiation in yeast.

The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.

Target size analysis by radiation inactivation: the use of free radical scavengers.

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.

Radiation inactivation analysis of sarcoplasmic reticulum Ca-ATPase in membrane-bound form and in detergent-solubilized monomeric states.

The sarcoplasmic reticulum Ca-ATPase was subjected to target size analysis by radiation inactivation in various buffer conditions and after solubilization in monomeric form in non-ionic detergent and in SDS. The target size was also determined for Ca-ATPase in bidimensional crystals formed in the presence of decavanadate or lanthanide. The standardization obtained with defined monomers of Ca-ATPase shows that the target size of Ca-ATPase in the functional membrane-bound state may be ascribed to a single peptide chain, possibly with surrounding lipid. Further analysis of the radiation inactivation sizes of various partial reactions of the pump cycle, including phosphorylation and Ca2+ occlusion, indicated much smaller values than the target size pertaining to decomposition of the whole peptide chain. This is consistent with the existence of separate functional domains within a single peptide chain.

Two UVC-induced stress response pathways in HeLa cells identified by cDNA microarray.

Environmental toxins induce multiple effects in vivo, involving various molecular pathways. The ultraviolet C (UVC, 254 nm) component of sunlight can cause strong cytotoxic and genotoxic effects. In this study, UVC-induced stress response factors were analyzed by cDNA microarray, using the Millennium(R) Nylon membrane chip system. HeLa cells were irradiated with 30 Joule/m(2)/sec UVC, incubated for 30 or 60 minutes and then subjected to the analysis. Multiple chips were used for each experimental condition so that the data could be analyzed statistically. Principal component analysis (PCA) was used to identify groups of genes whose expression changed in a similar manner with time post-UVC irradiation. Three major factors were identified, depending on the directionality of expression changes in each gene. The factor loadings in all three identified gene groups were high, indicating that genes within each group were highly correlated. Two factors exhibited significantly changed expression patterns after 30 minutes of incubation but in the opposite direction. This indicates that the "immediate early" UVC-induced stress response was elicited by two major pathways. Interestingly, expression of the genomic damage-inducible GADD genes, as well as p53, was initially decreased, unlike the "immediate early" genes Fos/Jun and Egr-1, which were strongly increased after 30 minutes of incubation. The results indicate that PCA used in the analysis of pre-hypothesized, functionally related genes can identify the potential subpathways in a group. This method provides a novel approach for identifying functionally-related genes in microarray studies.

Changes in lipid peroxide levels and activity of reactive oxygen scavenging enzymes in skin, serum and liver following UVB irradiation in mice.

The effect of acute UVB on the generation of reactive oxygen species (ROS) in the skin and the induction of ROS scavenging enzymes in situ was examined. Lipid peroxide levels and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and D-glucose-6-phosphate dehydrogenase (G-6-P-D) were determined in the skin, serum, and liver of ICR mice subjected to 1400 mJ/cm2 of acute UVB irradiation. In irradiated skin, lipid peroxides were increased at 3 and 24 hr after irradiation, whereas the four ROS scavenging enzymes were generally decreased during the first 48 hr after irradiation. In the serum, lipid peroxides showed an increase at 3 hr, but enzyme activities remained negligible. In the liver, lipid peroxides showed similar behaviour to that in skin. GSH-Px activity in the liver was decreased during the first 24 hr, whereas G-6-P-D showed substantial fluctuation and SOD and catalase activities showed no change. These data are consistent with a model in which lipid peroxides generated in the UVB-irradiated lesions are transported to the liver and there metabolized by the scavenging enzymes induced in situ.

[The hormonal status and carbohydrate-energy metabolism of rats after the long-term action of low doses of ionizing radiation and heat]. / Gormonal'nyi status i uglevodno-énergeticheskii obmen u krys posle dlitel'nogo deistviia malykh doz ioniziruiushchego izlucheniia i tepla.

Hormonal status (blood content of triiodothyronine, thyroxin, insulin, 11-hydroxycorticosteroids), dehydration in the Krebs cycle, and activity of the first enzyme in the pentose-phosphate cycle, glucose-6-phosphate dehydrogenase, in the brain and myocardium of white rats were studied at different time periods after separate and combined prolonged exposure to radiation in relatively small doses and heat. It was found that combination of ionizing radiation and heat led to hypofunction of the endocrine glands and inhibition of dehydration processes in the Krebs Cycle.

[Post-irradiation changes of metabolic and functional activity of adhesive macrophages]. / Postradiatsionnye izmeneniia metabolicheskoi i funktsionalnoi aktivnosti adgeziruiushchikh makrofagov.

Research into dose- and time-dependent alterations of quantitative and qualitative composition of the peritoneal cells has been carried out. Exposure at 3, 5, 7, or 9 Gy reduced the total number of peritoneal cells, increased the activity of the lactate-, malate-, and qlucoso-6-phosphate dehydrogenases, of the acid phosphatase and catepsin D; the amount of the cell protein in adhesing macrophages was also increased. The exposed macrophages showed enhanced phagocytic and cytotoxic activity as evidenced by the increase in luminol- and lucigenin-enhanced chemilumenscence in vitro. The most pronounced changes took place 7-9 days after exposure at 9 Gy.

[Radiation disorder of enzyme synthesis in the perinatal period of ontogeny]. / Issledovanie radiatsionnogo narusheniia sinteza fermentov v perinatal'nyi period ontogeneza.

A change of enzymatic differentiation in the rat liver during the perinatal developmental period after gamma-irradiation on the 7-9th and 19th days of embryogenesis in doses 0.5, 2 and 6 Gr has been shown on the example of glucose-6-phosphatase (G-6-P-ase) and tyrosine aminotransferase (TAT). The protein-synthesizing machinery was not damaged at these doses. The radiation inhibition of G-6-P-ase synthesis was relieved by the injection of thyroxine. A dependence was shown between the radiation increase of TAT activity and changes in cAMP system (increase of cAMP level, decrease of phosphodiesterase activity, intensification of response of adenylate cyclase complex to biogenic amines). A suggestion is put forward that the radiation damage of the enzymes under study is mediated by a change in the number of hormonal inductors.

[Effect of gamma radiation on glucose-6-phosphate dehydrogenase activity in the erythrocytes of healthy persons after submaximal physical exertion]. / Wplyw promieniowania gamma na aktywnosc dehydrogenazy glukozo-6-fosforanowej (G6P-DH) w krwinkach czerwonych ludzi zdrowych poddanych submaksymalnemu wysilkowi fizycznemu.

The authors studied effects of gamma radiation and submaximal physical exercise on G6P-DH activity in healthy men erythrocytes. Twelve men aged 20-22 were examined. They were loaded by physical exercise (at doses of 2 W/kg body weight) for 15 minutes. Erythrocytes were exposed to gamma radiation (500 Gy doses) from 60Co source. The activity of G6P-DH in erythrocytes was estimated by Boehringer-Mannheim test. Gamma radiation at 500 Gy dose was found to inhibit G6P-DH enzymatic activity in erythrocytes both at rest and after submaximal physical exercise. Furthermore, submaximal physical exercise was indicated to increase the G6P-DH activity in irradiated erythrocytes.

[Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation]. / Aktivnost' nekotorykh fermentov éritrotsitov u kurinykh émbrinov i tsypliat posle gamma-oblucheniia.

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.

Radiation target analysis of glycoproteins.

The radiation sensitivity of glycoproteins is shown to depend only on the protein portion of the molecule. An artificially created glycoprotein containing glucose-6-phosphate dehydrogenase observes this rule as well as natural enzymes and receptors containing from 2 to 50% carbohydrate. No exceptions have been found. Radiation damage to carbohydrates occurs close to the site of the primary ionization, with little spread of damage into attached polypeptides.

Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis.

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at -70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.

Electrophoretic variants of blood proteins in Japanese. IV. Prevalence and enzymologic characteristics of glucose-6-phosphate dehydrogenase variants in Hiroshima and Nagasaki.

Electrophoretic screening of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PD) was conducted one sample of 9,260 children born to the atomic bomb survivors in Hiroshima (Honshu) and Nagasaki (Kyushu). The prevalence of electrophoretic variants was 0.11% in males and 0.42% in females in Hiroshima, and 0.16% in males and 0.31% in females in Nagasaki. Enzymologic characteristics of 10 variants obtained from three males and seven hemizygous fathers of heterozygous females were examined. As a result, three new types of G6PD variants were identified among five variants detected in Hiroshima, and three new types among five variants in Nagasaki. All the variants except one belonged to Class 3, as defined by Yoshida et al. (1971).