Effect of nitric oxide donor (NaNO2) on the stability of non-transformed and malignant cells to ultraviolet and gamma-radiation

A decreased sensitivity of the Chinese hamster cells (line V-79) to gamma-radiation under the influence of nitric oxide induction was shown elsewhere. This effect is connected hypothetically with post-radiation reparation of DNA. The investigation of the nitric oxide donor effect on sensitivity of these to UV-radiation is of interest, because this radiation is an important ecological factor of the environment. The question of retention of nitric oxide positive effect on UV and gamma-radiation sensitivity in malignant HeLa cells is no less actual, because these cells significantly differ from normal cells of line V-79. We demonstrated that the donor of nitric oxide enhances stability of the Chinese hamster cells (line V-79) to UV-radiation, as well as to gamma-radiation independently of the time of cell incubation with sodium oxide donor before or after irradiation. The inefficiency of nitric oxide as a factor increasing UV-stability of cells was shown for malignant HeLa cells. A 1 h long incubation of these cells with NO-donor before gamma-irradiation decreased the number of chromosome aberrations, and conversely, the addition of this agent to the HeLa cell culture after gamma-irradiation did not change the radiostability. It may be inferred that distinctions in behaviour of nitric oxide in cultures of V-79 and HeLa cells using UV-radiation may be explained by transformation of the latter special features of their damage, and by the following reparation.     

The formation of DNA-protein cross-links in response to gamma radiation, UV irradiation and chemical agents]

The present paper deals with the damages that are induced by numerous agents, such as gamma-radiation, UV-radiation, visible fluorescent light radiation, and a wide range of chemical agents, and lead to the formation of DNA-protein cross-links. This is the most unknown and obscure type of damage to cells. It is, however, well known that these damages are reparable.     

Loss of Gadd45a does not modify the pulmonary response to oxidative stress

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.     

Survival and recovery of yeast cells after combined treatment with ionizing radiation and heat

Cell survival, recovery kinetics and inactivation forms after successive and simultaneous treatments with gamma rays (60Co) and high temperatures were studied in diploid yeast cells capable of recovery. Both the extent and the rate of the recovery were shown to be greatly decreased with increase in the duration of heat treatment (60 degrees C) followed by radiation and with increase in exposure temperature after simultaneous treatment with heat and radiation. A quantitative approach describing the recovery process was used to estimate the probability of recovery per unit time and the irreversible component of damage after the combined treatment with heat and radiation. It was shown that the probability of recovery was independent of the conditions of the treatment with heat and radiation, while the irreversible component gradually increased as a function of the duration of heat treatment (60 degrees C) after sequential treatment with heat and radiation and as a function of the exposure temperature after simultaneous treatment with heat and radiation. The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division. It is concluded on this basis that the synergistic interaction of ionizing radiation and hyperthermia in yeast cells is not related to the impairment of the recovery capacity itself and that it may be attributed to an increased yield of irreversible damage.     

Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

UV-B light induces an adaptive response to UV-C exposure via photoreactivation activity in Euglena gracilis.

Phytoplankton such as Euglena are constantly exposed to solar light which is used for photosynthesis. Although the solar ultraviolet (UV) induces DNA damage such as cyclobutane-pyrimidine dimers (CPDs), many kinds of living organisms can repair CPDs by photoreactivation (PR) utilizing the near-UV/blue light component in sunlight. Euglena cells are known to possess such PR activity. In the present paper, the formation of CPDs induced by UV-C exposure and the photoreactivation PR repair of these CPDs by UV-A are demonstrated. To clarify the adaptive responses prior UV-B irradiation on PR activity, cells were cultured in the dark or under UV-B light. When the cells were cultured in the dark for 3 d prior to UV-C exposure, PR activity decreased. When the cells were cultured under UV-B light, however, PR activity increased. These results suggest that exposing the cells to UV-B prior to exposure to UV-C induced an adaptive response towards DNA damage caused by UV-C exposure, and this UV-C induced damage was repaired through PR activity.     

Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer

Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60J/m(2). NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

Prognosis of yeast cells recovery after simultaneous exposure to UV-radiation and hyperthermia

The results of experimental investigations of survival of diploid yeast cells Saccharomyces cerevisiae (strain XS800) after simultaneous exposure to UV-radiation (254 nm) and hyperthermia (53-57 degrees C) have been described. It was shown that the portion of cells capable of recovery in innutrient medium after the action of these agents decreased with the increasing of temperature under which the irradiation was occurred. Mathematical model taking into account the synergistic interaction was suggested for quantitative prediction of irreversible component after combined actions of these agents. A good correspondence between experimental data and model predictions has been demonstrated. The importance of the results obtained for the interpretation of the synergistic interaction mechanisms are discussed.     

Protein synthesis and the recovery of both survival and cytoplasmic “petite” mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) and chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the ϱ⁺ genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in statonary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) processes for the ϱ⁻ induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis.When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressedor depressed state of the mitochondria.     

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).     

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).     

Factors influencing survival of mammalian cells exposed to hypothermia. V. Effects of hepes, free radicals, and H2O2 under light and dark conditions

Cytotoxicity resulting from the interaction of fluorescent light from a flow hood with Hepes-buffered cell culture medium at room temperature was demonstrated. Toxicity was prevented by keeping both cells (V79 Chinese hamster) and medium shielded from direct fluorescent light ("dark conditions") or by supplementing the medium with 10 micrograms/ml catalase; this suggests that extracellular hydrogen peroxide is a major cause of the lethal effect under "lighted conditions." No sensitization resulted from the exposure of cells in a sodium bicarbonate (SBC)-buffered medium to fluorescent light, nor in a catalase supplemented SBC-buffered medium. The Hepes/light reaction during routine cell manipulations presensitized cells to hypothermia damage in the dark with the presensitization being more severe for 5 than for 10 degrees C hypothermic exposure. Presensitization was prevented by performing the complete experiment under dark conditions or by supplementing the medium with 10 micrograms/ml catalase. However, catalase did not improve the hypothermic survival when experiments were performed under dark conditions. Hence, 10 micrograms/ml catalase does not protect cells from hypothermic (5 and 10 degrees C) damage per se, but rather from Hepes/light sublethal damage which interacts with hypothermic sublethal damage to result in lethal lesions. Additionally, under dark conditions, superoxide dismutase (SOD), allopurinol, catalase plus SOD, DMSO, or mannitol did not improve survival when present during hypothermic storage, suggesting that extracellular superoxide anion, hydrogen peroxide, or hydroxyl radicals are not the cause of cell killing under conditions of pure hypothermia uncomplicated by prehypothermic ischemia or hypoxia.     

UV-screening of grasses by plant silica layer?

UV-screening by terrestrial plants is a crucial trait since colonization of terrestrial environments has started. In general, it is enabled by phenolic substances. Especially for grasses it remains unclear why plants grown under the absence of UV-B-radiation exhibit nonetheless a high UV-B-screening potential. But this may be explained by the UV-screening effect of the silicon double layer. It was shown for seedlings of soybeans (Glycine max L.) and wheat (Triticum aestivum L.) that enhanced silicon supply reduces stress induced by UV-radiation. Even more important is a direct correlation between silicon content in the epidermis near area (intercellular spaces) and the absorption of UV-radiation in this area shown in other papers. The silicon double layer may act like a glass layer and decreases the transmission of UV-radiation at the epidermis near area. In summary, the absorbance/reflection of ultraviolet radiation is dependent on the characteristics of the epidermis near area of leaves, particularly the occurrence (qualitatively and quantitatively) of phenolic substances and/or a silicon double layer in this area. Consequently, UV-screening by plant silicon double layer should get more attention in future research with emphasis on effects of UV-radiation on plant physiology.     

PHOTOINHIBITION OF PHOTOSYNTHESIS IN ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1, 2

A laboratory study was made of the inhibition of photosynthesis, as measured by the radiocarbon method, of Asterionella Formosa Hass. by light from tungsten halogen lamps of irradiances up to that of full sunlight. The observed inhibitions were of total fixation of carbon and were not due to greatly increased release of extracellular products of photosynthesis. Inhibition increased width irradiance and with time of exposure. It was greater at high temperature, at high oxygen concentrations and when the cells were nutrient deficient. Recovery from exposure to high irradiances took place both in the dark and at low irradiances. The inhibition, which is similar to that observed under natural conditions, has the characteristics of photooxidative damage to both photochemical and dark reaction mechanisms.     

Caffeine and artificial insemination

We studied the reactivation of cells and the repair of photomutagenic damage induced by xanthotoxin and visnagin plus NUV in arg-1 cells of Chlamydomonas reinhardii. Maintenance of liquid cultures in the dark resulted only in a slight reactivation of cells, even after 24 h. Repair of photomutagenic damage was more efficient: within 24 h the number of Arg⁺ revertants was reduced by 50% in cells cultured in the dark at 20°C. The repair was more efficient at 30°C. At the beginning of dark cultivation an after-effect could be observed. Cultivation in standard white light instead of dark after treatment resulted in a very strong after-effect. Therefore it was not possible to detect any photoreactivation.After treatment with xanthotoxin plus standard white light (24 h) neither reactivation of cells nor repair of photomutagenic damage was found. The after-effect was higher than after xanthotoxin plus NUV. It is possible that a small amount of repair could be masked by the after-effect.Treatment with visnagin yielded similar results. The photomutagenic effect of visnagin is described for the first time in this paper. The drug is a much less effective photomutagen than xanthotoxin. The photomutagenesis of visnagin may be attributable to photoproducts similar to those formed after treatment with furocoumarins.No definite conclusion can be drawn from the present results regarding the basis for the observed lack of repair (or reduced repair) after standard white light treatment; a possible cause might be a preferential formation of bi-adducts under these conditions.     

Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria

In temperate lakes, asynchronous cycles in surface water temperatures and incident ultraviolet (UV) radiation expose aquatic organisms to damaging UV radiation at different temperatures. The enzyme systems that repair UV‐induced DNA damage are temperature dependent, and thus potentially less effective at repairing DNA damage at lower temperatures. This hypothesis was tested by examining the levels of UV‐induced DNA damage in the freshwater crustacean Daphnia pulicaria in the presence and absence of longer‐wavelength photoreactivating radiation (PRR) that induces photoenzymatic repair (PER) of DNA damage. By exposing both live and dead (freeze‐killed) Daphnia as well as raw DNA to UV‐B in the presence and absence of PRR, we were able to estimate the relative importance and temperature dependence of PER (light repair), nucleotide excision repair (NER, dark repair), and photoprotection (PP). Total DNA damage increased with increasing temperature. However, the even greater increase in DNA repair rates at higher temperatures led net DNA damage (total DNA damage minus repair) to be greater at lower temperatures. Photoprotection accounted for a much greater proportion of the reduction in DNA damage than did repair. Experiments that looked at survival rates following UV exposure demonstrated that PER increased survival rates. The important implication is that aquatic organisms that depend heavily on DNA repair processes may be less able to survive high UV exposure in low temperature environments. Photoprotection may be more effective under the low temperature, high UV conditions such as are found in early spring or at high elevations.     

紫外辐射对两种体色型麦长管蚜生物学特征的影响

紫外辐射对蚜虫产生了强烈的选择压力,可导致蚜虫种下遗传分化,而紫外辐射对蚜虫种下体色分化的影响尚不清楚.本研究选定两种不同体色型的麦长管蚜初生若蚜经过30 W紫外线（UVB波）照射5 d,每天30 min,在不同温度（15 ℃、20 ℃和25 ℃）和不同小麦品种（小偃-22、Astron）条件下,通过在人工智能控制温室内单头饲养,分别测定了各处理麦长管蚜的发育历期、体质量差及相对日均体质量增长率等生物学参数.结果表明：在品种小偃-22上,温度较低时,紫外辐射显著延缓了绿色型麦长管蚜生长,而温度较高时,紫外辐射显著延缓了红色型麦长管蚜生长;在品种Astron上,温度较低时,紫外辐射显著延缓了绿色型麦长管蚜生长,而温度较高时,紫外辐射对两种体色型麦长管蚜生长均无显著影响.说明两种体色型蚜虫对紫外胁迫的反应不同,且紫外胁迫对麦长管蚜的影响与温度和小麦品种有关.