Effect of hyperthermia on the repair of sublethal radiation damage in normal and membrane fatty acid substituted fibroblasts

The interaction of heat and X irradiation was studied in normal and polyunsaturated fatty acid (PUFA) substituted mouse fibroblast LM cells. As a result of the substitution the membranes of the PUFA cells were more fluid than the membranes of the normal cells. Three different heat doses were applied (60 min 42 degrees C, 20 min 43 degrees C, and 10 min 44 degrees C) in combination with single or split doses of X rays. Heat radiosensitization was the largest for the 60 min 42 degrees C treatment. Heat radiosensitization and the heat-induced inhibition of the rate of sublethal damage repair were the same for the normal and the PUFA cells. It is concluded from the experiments reported that the processes of hyperthermic inhibition of SLD repair and hyperthermic radiosensitization are independent of membrane fluidity and membrane fatty acid composition.     

DNA damage and repair in epithelial (mucous) cells and crypt cells from isolated colon

Colon epithelium is made up of two general classes of cells, surface cells which are post-mitotic and crypt cells which contain the proliferative population. Their relative vulnerability to environmental damage and ability to perform DNA repair are important issues in colon carcinogenesis. DNA damage and repair was studied by the nucleoid sedimentation method in freshly isolated crypt cells for comparison with previous studies of post-mitotic surface epithelial cells. Suspensions of crypt cells were isolated from preparations of mouse colon by a series of sequential incubations in buffer containing 1.5 mM EDTA. Treatment of crypt cells for 30 min with 1.2 X 10(-6) M methyl methane sulfonate (MMS), photoaffinity labeling with 1 X 10(-6) M ethidium monoazide, lithocholic acid (2.5 X 10(-4) M) treatment for 1 h or X-irradiation at 1500 rads resulted in single-strand breaks in the DNA, which were repaired after 2 h of additional incubation. Interestingly, X-rays at 1000 rads and lithocholic acid (LA) (2.5 X 10(-6) M) after 30 min incubation failed to produce the detectable shift in nucleoid sedimentation characteristic of single-strand breaks, perhaps due to rapid repair by these proliferative cells. UV-irradiation failed to provoke strand incision as was also observed for the superficial post-mitotic cells in the previous studies. These studies showed the feasibility of studying DNA damage and repair processes in these two classes of colon epithelial cells in response to specific carcinogenic insult.     

Antimetabolite sensitivity and magnesium uptake by thermally stressed Vibrio parahaemolyticus

Metabolic inhibitors were added to a culture medium inoculated with theramlly stressed Vibrio parahaemolyticus to obtain information pertaining to biosynthetic processes required for recovery from heat damage. Ribonucleic acid and protein syntheses, in addition to membrane repair, were required during recovery of injured cells. Neither nucleic acid nor Mg2+ leakage was noted to occur during the time cells were subjected to heat stress. Studies revealed that Mg2+ was apparently taken up by cells of V. parahaemolyticus during the first 30 min after thermal treatment, indicating a possible increased requirement for Mg2+ for membrane and/or ribosome stability and repair.     

Antimetabolite sensitivity and magnesium uptake by thermally stressed Vibrio parahaemolyticus.

Metabolic inhibitors were added to a culture medium inoculated with theramlly stressed Vibrio parahaemolyticus to obtain information pertaining to biosynthetic processes required for recovery from heat damage. Ribonucleic acid and protein syntheses, in addition to membrane repair, were required during recovery of injured cells. Neither nucleic acid nor Mg2+ leakage was noted to occur during the time cells were subjected to heat stress. Studies revealed that Mg2+ was apparently taken up by cells of V. parahaemolyticus during the first 30 min after thermal treatment, indicating a possible increased requirement for Mg2+ for membrane and/or ribosome stability and repair.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

A simple technique for quantitation of low levels of DNA damage in individual cells

Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.     

Characterization of Injury Incurred by Escherichia coli upon Freeze-Drying

When cells of Escherichia coli ML30 were suspended in 2% gelatin and frozen at -40 C, no appreciable metabolic damage or death occurred. After freeze-drying for 8 hr at a platen temperature of 49 C and rehydration with a mineral salts medium, survival of the cells was 0.6%. Metabolic damage of the survivors was found to be 23%. Permeability alterations were detected by several criteria. Freeze-dried cells were susceptible to antibiotics normally ineffective against E. coli and leakage of ribonucleic acid (RNA) occurred. Analysis of ribosomal extracts of rehydrated freeze-dried cells demonstrated the presence of appreciable degradation products. Permeability alterations were shown to be reversible by the observation that antibiotic susceptibility was a time-dependent process and that the gratuitous inducer of beta-galactosidase was not concentrated by freeze-dried cells until the injured cells had been incubated in a nutrient medium for 300 min or more. At approximately the same time, metabolic damage was repaired. RNA synthesis preceded protein synthesis by about 150 min, and deoxyribonucleic acid synthesis occurred with the resumption of normal growth. This was interpreted to be the result of repair of RNA taking place before protein synthesis and growth could resume. A pronounced increase in the lag time of freeze-dried cells was also observed. Peptides and Casamino Acids shortened the lag time for freeze-dried cells but not for the controls. Glycerol and glucose were found to be better carbon sources for growth of freeze-dried cells than sodium lactate or sodium succinate.     

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.     

Effect of melanin on radiation response of CHO cells

The effect of the presence of melanin on the response of mammalian cells to ionizing radiation was investigated in a model system utilizing the ability of Chinese hamster ovary cells to incorporate melanin by endocytosis. Cells were incubated in monolayer cultures from 2 to 20 hours with melanin prepared from 'beef eye' or synthesized by air oxidation of 3,4-dihydroxyphenylalanine. For asynchronous cultures, the survival curve parameters for cells incubated with both types of melanin were indistinguishable from those of the same cells without added melanin. The radiation response to fractionated doses of 6 Gy separated by various periods did not indicate any effect of melanin on the extent or kinetics of repair of sublethal damage. Likewise, the repair of potentially lethal damage in plateau phase cultures was unaffected by the presence of melanin. Thus the explanation for the clinical radiation resistance of melanomas in the absence of a direct radiation effect might more likely be found in consideration of other factors such as the role of melanin in oxygen consumption or in differentiation.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

The effect of chromatin decondensation on DNA damage and repair

The effects of chromatin compaction on X-radiation-induced cell killing and the induction and repair of DNA damage were studied in Chinese hamster ovary cells deprived of isoleucine for 24 h (Ile- cells) and compared to untreated controls. The results show that chromatin is decondensed in Ile- cells; i.e., in Ile- cells the nuclear area occupied by heterochromatin decreased 30-fold over control cells, both the rate and limit of micrococcal nuclease digestion were greater for Ile- cells, and 14.2% more propidium iodide was intercalated into the Ile- cell chromatin. The X-ray-induced cytotoxicity did not change in Ile- cells versus the control cells (D0 = 0.99 Gy) nor did the X-ray-induced DNA damage. However, the repair of DNA damage produced by 10 Gy proceeded with different kinetics in Ile- cells when compared to the controls. The initial rate of DNA damage repair was slower in Ile- cells by a factor of 2 compared to controls (the time required to rejoin 50% of the lesions was 6 versus 3 min, respectively). However, after 2 h of repair no DNA damage was detected in either group. Therefore, we conclude that this decondensation of chromatin, per se, does not directly modify the induction or ultimate repair of DNA damage by X radiation or cell clonogenicity and thus does not appear to be a primary factor in cell survival.     

The saturated repair kinetics of Chinese hamster V79 cells suggests a damage accumulation--interaction model of cell killing

The aim of this study is to determine whether the repair process in log-phase Chinese hamster V79 cells exposed to X rays is unsaturated, saturable, or saturated. The kinetics of recovery from damage induced by 2 to 14 Gy of 250 kVp X rays was studied by treating cells with 0.5 M hypertonic saline for 20 min at different postirradiation repair intervals. From the kinetic data, the repair half-time (t1/2), the repair time (time needed to attain maximal survival), and the recovery ratio were calculated. The results show that the t1/2 (1.42 min/Gy) and the repair time (6.04 min/Gy) increase linearly with dose, the logarithm of the recovery ratio increases linear-quadratically with dose, and the D0 increases linearly with repair interval at a rate of 2.4 cGy/min. From these results we suggest a model: the repair of damage (undefined lesions) necessary for cell survival is effected by a repair process (t 1/2 of 1.42 min/Gy) which is saturated at doses as low as 2.4 cGy; repair saturation leads to a dose-dependent accumulation of repairable lesions; and interaction among accumulated repairable lesions results in the induction of irreparable (lethal) lesions. We call this the accumulation-interaction model of cell killing by low-LET radiation.     

Exposure of mammalian cells to 60-Hz magnetic or electric fields: analysis of DNA repair of induced, single-strand breaks

DNA damage was induced in isolated human peripheral lymphocytes by exposure at 5 Gy to 60Co radiation. Cells were permitted to repair the DNA damage while exposed to 60-Hz fields or while sham-exposed. Exposed cells were subjected to magnetic (B) or electric (E) fields, alone or in combination, throughout their allotted repair time. Repair was stopped at specific times, and the cells were immediately lysed and then analyzed for the presence of DNA single-strand breaks (SSB) by the alkaline-elution technique. Fifty to 75 percent of the induced SSB were repaired 20 min after exposure, and most of the remaining damage was repaired after 180 min. Cells were exposed to a 60-Hz ac B field of 1 mT; an E field of 1 or 20 V/m; or combined E and B fields of 0.2 V/m and 0.05 mT, 6 V/m and 0.6 mT, or 20 V/m and 1 mT. None of the exposures was observed to affect significantly the repair of DNA SSB.     

Genetic Analysis of Repair of Ultraviolet Damage by Competent and Noncompetent Cells of Bacillus subtilis

The repair of ultraviolet (UV) damage in Bacillus subtilis W23T(-) has been studied by transformation with deoxyribonucleic acid (DNA) extracted from irradiated cells before and after repair. The extent of repair of genetic markers by donor cells after low or moderate doses of UV was found to be related only to the initial degree of inactivation. After a very high dose, further inactivation occurred, also in proportion to initial damage. In addition, the competent recipient cells were shown to repair approximately 75% of the damage in transforming DNA. The sensitivities of markers irradiated either in vivo or in vitro appeared to be related to map position, the more proximal markers showing a greater resistance to UV inactivation.     

A calmodulin antagonist has no effect on the repair of X-ray-induced damage in a murine mammary carcinoma cell line

The effects of the calmodulin antagonist W13 were determined on potentially lethal damage repair, sublethal damage repair, and X-ray-induced DNA damage repair following X irradiation of 67 murine mammary carcinoma cells in the proliferative and quiescent states. Studies with W13 (20 micrograms/ml) on proliferating cells showed that the cells rounded up within 2 h but stayed attached to the dishes and there was a slight transient G2 block by 6 h. Also, the proportion of S-phase cells at 12 h was reduced to 65% of control with the concurrent [3H]thymidine incorporation reduced to 62% of control. There was no detectable effect from this pharmacological dose of W13 either on PLDR in proliferating cells at 400 and 800 rad or on quiescent cells at 200 and 400 rad. Likewise, there was no measurable effect on SLDR in either proliferating or quiescent cells at equally split doses of 800 and 600 rad, respectively. In addition, for control vs W13-treated proliferating cells, no difference was detected either in the induction of DNA damage by X irradiation or in the initial rate of repair (T 1/2 approximately equal to 7 min), as measured by the alkaline filter elution assay. In contrast to uv and bleomycin-induced damage, these data suggest that calmodulin may have no major role in either the molecular or cellular recovery from X-ray-induced damage in mammalian cells.     

Induction and subsequent repair of DNA damage by fast neutrons in cultured mammalian cells

The induction and repair of DNA damage were studied by a DNA unwinding method in mouse L5178Y cells exposed to fast neutrons. DNA lesions induced by fast neutrons were classified into three types from their repair profiles: fast-reparable breaks (T1/2 = 3-5 min), slow-reparable breaks (T1/2 = 70 min), and nonreparable breaks. The repair rates of both fast-reparable and slow-reparable breaks were almost the same as those of corresponding damage induced by low-LET radiation. Neutrons induced a smaller amount of fast-reparable damage, an almost equal amount of slow-reparable damage, and a larger amount of nonreparable damage than those induced by equal doses of gamma rays or X rays. RBEs for fast- and slow-reparable damage were 0.3 and 0.9, respectively. The RBE for nonreparable damage was dose dependent and was 1.4 at the level of 100 breaks/10(12) Da DNA. Among the three types of lesions, only the nonreparable damage levels correlated with the linear-quadratic shape of the survival curves and with the enhanced killing effectiveness of neutrons (RBE = 1.7 at D0).     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

In vivo non-enzymatic repair of DNA oxidative damage by polyphenols

The non-enzymatic repair of DNA oxidative damage can occur in a purely chemical system, but data show that it might also occur in cells. Human hepatoma cells (SMMC-7721) and human hepatocyte cells (LO2) were treated with 200 μM H₂O₂ for 30 min to induce oxidative DNA damage quantified by amount of 8-OHdG and degree of DNA strand breaks, without inducing enzymatic repair. The dynamics of enzymatic repair activity quantified by unscheduled DNA synthesis, within 30 min after removal of H₂O₂ enzymatic repair mechanism has not been initiated. However, pre-incubation with low micromolar level polyphenols, quercetin or rutin can significantly attenuate DNA damage in both cell lines, indicating that the polyphenols did not work through an enzymatic mechanism. Unscheduled DNA synthesis after removal of H₂O₂ was also markedly decreased by quercetin and rutin. Combined with our previous studies of fast reaction chemistry, the inhibitory effect of polyphenols have to be assigned to non-enzymatic repair mechanism rather than to enzymatic repair mechanism or antioxidant mechanism.     

Overview of the spaceflight radiation environment and its impact on cell biology experiments

Variables studied in typical cellular radiation biology experiments are cell killing, mutagenesis, transformation to malignancy, heritable damage, and DNA damage and repair. Dose response curves for cells exposed to low-LET radiations and some high LET radiations are well known. The low-LET dose rate in low earth orbit is roughly 1.0 mSv/day, the heavy-ion (Z>2) flux is about 1.0 particle/cm2-s corresponding to about 0.3 mSv/day, and the integrated neutron flux is roughly 2 neutrons/cm2-s corresponding to 0.012 mGy/d or, assuming a QF of 10, 0.12 mSv/d. Published dose-response curves were used to estimate the probability that a mammalian cell will be affected by each of the above types of damage. As a general approximation the exposure of an experimental cell population to the space radiation environment for 100 days will result in the following probabilities of damage per cell: cell killing based on clonogenicity 0.02, mutagenesis per locus based on phenotype analysis 1 x 10(-6), point mutation induction 2 x 10(-8) per locus, malignant transformation in vitro based on colony morphology 1.2 x 10(-5), heritable damage based on colony size 0.02, and induced DNA double-strand breaks based on fragment analysis by electrophoresis 3.5/cell or 0.26/cell after repair. Most of these figures are accurate to within a factor of 2. Thus the spaceflight radiation environment has essentially undetectable impact on typical cell biology experiments unless experimental goals involve the precise measurement of one of the above end-points. Other in vitro end-points, such as tissue morphogenesis and cell differentiation, are expected to be similarly unaffected by the spaceflight radiation environment.