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.

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.

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.

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.

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.

Effects of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae).

Stipa capillata (Poaceae) seeds were harvested from a control area (displaying a gamma dose rate of 0.23 micro Sv h(-1)) (C plants) and from two contaminated areas (5.4 and 25 micro Sv h(-1)) on the Semipalatinsk nuclear test site (SNTS) in Kazakhstan. The plants were grown for 124 d in a greenhouse under controlled conditions and exposed to three different treatments: (0) control; (E) external gamma irradiation delivered by a sealed 137Cs source with a dose rate of 66 micro Sv h(-1); (E+I) E treatment combined with internal beta irradiation due to contamination by 134Cs and 85Sr via root uptake from the soil. The root uptake led to a contamination of 100 Bq g(-1) for 85Sr and 5 Bq g(-1) for 134Cs (of plant dry weight) as measured at harvest. The activity of SOD, APX, GR, POD, CAT, G6PDH, and MDHAR enzymes was measured in leaves. Under (0) treatment, all enzymes showed similar activities, except POD, which had higher activity in plants originating from contaminated areas. Treatment (E) induced an enhancement of POD, CAT, GR, SOD, and G6PDH activities in plants originating from contaminated areas. Only control plants showed any stimulation of APX activity. Treatment (E+I) had no significant effect on APX, GR, CAT, and POD activities, but MDHAR activity was significantly reduced while SOD and G6PDH activities were significantly increased. The increase occurred in plants from all origins for SOD, with a greater magnitude as a function of their origin, and it occurred only in plants from the more contaminated populations for G6PDH. This suggests that exposure to a low dose rate of ionizing radiation for almost a half century in the original environment of Stipa has led to natural selection of the most adapted genotypes characterized by an efficient induction of anti-oxidant enzyme activities, especially SOD and G6PDH, involved in plant protection against reactive oxygen species.

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation.

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.

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.

Studies of methyl 2-nitroimidazole-1-acetohydroxamate (KIN-804) 1: effect on free radical scavenging system in mice bearing Ehrlich ascites carcinoma.

Methyl 2-nitroimidazole-1-acetohydroxamate (KIN-804) is a 2-nitroimidazole derivative containing a hydroxamate side chain designed to enhance the radiosensitization response of hypoxic cells. The possible sensitization of tumor tissue by KIN-804 can be evaluated through investigation of the levels of the free radical scavengers; namely, glutathione (GSH) and its complex enzyme system including glutathione reductase (GR) and glutathione peroxidase (GSH-Px), as well as glucose-6-phosphate dehydrogenase (G-6-PD). Female albino mice were inoculated with Ehrlich carcinoma in the thigh. Administration of KIN-804 (i.p. 80 mg/kg body weight) was carried out 20 min before localized irradiation of 10 Gy. The data revealed that KIN-804 administration, followed or not by gamma irradiation, resulted in a significant decrease in GSH content in tumor tissues associated with inhibition in GR and G-6-PD activities. Blood GSH-Px was enhanced in tumor inoculated mice and the administration of KIN-804 returned it to the normal value. These changes were more noticeable in tumor bearing mice exposed to both KIN-804 and irradiation.

Radiation effects on the native structure of proteins: fragmentation without dissociation.

Several proteins (avidin, carboxypeptidase B, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, maltase, and peroxidase) composed of one to six subunits were irradiated in the frozen state. Each irradiated protein was examined by size-exclusion chromatography (SEC) and by denaturing gel electrophoresis (SDS-PAGE). All these proteins eluted from SEC as a single peak even though SDS-PAGE showed cleavage of the polypeptide backbone of the monomers. Thus, fragmentation of the subunits did not result in dissociation of the oligomeric structure.

[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.

Effect of environmental conditions on radiation target size analyses.

Target size determinations from radiation inactivation of proteins is dependent on the physical and chemical environment of the sample during radiation exposure. Effects of temperature and physical state have already been described. Buffers, the effects of protein concentration, and the addition of small molecules are examined for several enzymes. Phosphate buffer is found to have major effects on the rate of inactivation of certain, but not all, proteins. The amount of protein in irradiated samples is significant for all enzymes studied; the nature of the specific protein used is unimportant. Neither sucrose nor other glycitols could substitute for protein in target size determinations. Certain small molecules, especially cysteamine, were effective in sparing the need for high protein levels in radiation inactivation studies of four enzyme systems.

[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.

Target size analysis of prostaglandin endoperoxide synthase. Radiation inactivation of both cyclooxygenase and peroxidase correlated with the monomer of 72 kDa.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.

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.

Direct effects of radiation on the avidin-biotin system. Absence of energy transfer.

Frozen solutions of biotinylated glucose-6-phosphate dehydrogenase and fluorescently tagged avidin were exposed to high energy ionizing radiation. Parallel experiments with peroxidase coupled to streptavidin and with biotinylated phycoerythrin were also performed. The loss of function of each compound was analyzed according to target theory. Target analysis revealed that the radiation-sensitive mass associated with the enzymatic activity and that associated with the fluorescence were unchanged by irradiation in the strongly coupled state. Therefore the noncovalent bonds between biotin and avidin do not permit the transfer of radiation-deposited energy in amounts sufficient to destroy the activity of apposing molecule.

Glucose-6-phosphate dehydrogenase activity in CaNT tumours of different volume and response to tumour clamping and X-irradiation.

Changes in the levels of glucose-6-phosphate dehydrogenase (G6P-DH) activity, versus tumour volume were measured in vivo under normoxic conditions in the CaNT tumours grown in CBA mice. A monotonically increasing relation was found. Artificially induced tumour hypoxia resulting from 15 min of clamping was accompanied by enhanced G6P-DH activity, but one hour after release of the clamps (reflow), the specific activity of the enzyme decreased to about the same level as that of the normoxic controls. Also the levels of G6P-DH activity in normoxia were measured one hour after 5, 10 and 15 Gy of X-rays (100 kVp). A significantly higher G6P-DH activity which increased with dose, was found in the tumours. This paper gives evidence of increased G6P-DH activity linked with tumour progression, under gradual amplification of the metabolic unbalance. The augmentation of G6P-DH following X-irradiation probably represents a cellular adaptive defence mechanism during the demand for increased metabolism.

Functional size of acyl coenzyme A:diacylglycerol acyltransferase by radiation inactivation.

Rat liver acyl coenzyme A:diacylglycerol acyltransferase, an intrinsic membrane activity associated with the endoplasmic reticulum, catalyzes the terminal and rate-limiting step in triglyceride synthesis. This enzyme has never been purified nor has its gene been isolated. Inactivation by ionizing radiation and target analysis were used to determine its functional size in situ. Monoexponential radiation inactivation curves were obtained which indicated that a single-sized unit of 72 +/- 4 kDa is required for expression of activity. The size corresponds only to the protein portion of the target and may represent one or several polypeptides.

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction.

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.

Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions.

Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.