PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

The dependence of DNA repair induced by genetically hazardous exposures on the ionic strength of the medium containing the cells]

The dependence of UV-induced unscheduled DNA synthesis (UDS) in non-stimulated lymphocytes of human peripheral blood on the ionic strength (mu) of the culture medium has been shown. With the level of mu lower or higher than physiological (mu ph) the UDS significantly decreases. The effect of modification of mu due to changes in ionic strength is absent in the lymphocytes of patients with the classic form of xeroderma pigmentosum. This phenomenon may become useful for development of a new test revealing cells with genetically or physiologically changed system of UV-induced DNA repair. Mechanisms of investigated phenomenon, particularly their dependence on the chromatin structure, as well as the influence of ionic strength on binding the repair enzymes with DNA are discussed.     

DNA-repair capacity and lipid peroxidation in chronic alcoholics

The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p less than 0.01) and LPO in plasma was significantly higher (p less than 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = -0.078 x vit. C + 1.9; p less than 0.05) and between UDS and LPO values (UDS = -0.384 x LPO + 4.1; p less than 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Poly(ADP-ribose)-synthesis and excision repair in light sensitive skin disorders

Several data suggest a relationship of poly(ADP-ribose) (PAR) synthesis to DNA repair and the influence of some trace elements on the semiconservative and unscheduled DNA synthesis (UDS). Previously we found certain alterations in the UV-light induced UDS and in the contents of trace elements in the lymphocytes of patients with light sensitive skin disorders. In the recent study in polymorphic light eruption, cutaneous porphyrias and xeroderma pigmentosum the PAR synthesis and zinc, copper and manganese contents in the chromatin of the lymphocytes (measured by neutron activation analysis) were investigated. UV induced PAR synthesis was generally lower in the cells of polymorphic light eruption and especially in xeroderma pigmentosum with a reduced repair capacity whereas in cutaneous porphyrias no difference was observed. Some correlations occurred between the contents of trace elements studied and UDS as well in each group tested. It seems that PAR investigations throw new light upon our understanding of the pathomechanism of photodermatoses.     

Experimental assessment of embryotoxic action of low intensity ionizing irradiation in different periods of intrauterine development

The prolonged ionizing radiation exposure at the absorbed doze rate of 2.6 mGy/hour and the total accumulated doze of about 20 cGy during different periods of the intrauterine development caused disorders of a coordinated development of organs and the decrease of endurance in physical activity in the postnatal ontogenesis of the irradiated posterity. The results obtained are comparable to the consequences of irradiation exposure of the fetus at the accumulated doze of 125 cGy during all stages of pregnancy and give grounds for considering radiation exposure at the above-mentioned rate (20 cGy) as a biologically significant factor.     

Comparison of UV-induced unscheduled DNA synthesis in lymphocytes exposed to low doses of ionising radiation in vivo and in vitro

In an attempt to understand and ascertain the stimulatory effects of low-dose ionising radiation, a study was conducted to compare the changes in the UV-induced repair capacity of human blood cells exposed to low conditioning doses of ionising radiation under in vivo and in vitro conditions. A significant increase in the rate of UV induced Unscheduled DNA synthesis (UDS) in lymphocytes pre-exposed to low doses of ionising radiation was observed both under in vitro and in vivo conditions. There was also a significant correlation between the adapting dose and net UDS in lymphocytes of radiation workers implying that the triggering action of the adaptation process is dose dependent. However, on comparing the extent of UV-induced UDS of the in vivo and in vitro exposures, significantly higher rates of UDS were observed in the lymphocytes of radiation workers when compared to a corresponding in vitro adapting dose. We postulate that the response in vivo is much more pronounced due to cell repopulating events and extra cellular secretory factors like hormones etc,.     

Delayed effect of prenatal irradiation on the critical organs functional state in children after Chernobyl accident

Some indexes of a metabolism in 186 children exposed in utero through the Chernobyl accident were investigated ten years after the irradiation. All the children (basic and control groups) live in the territory with contamination less than 15 Ci/km2. It was shown, that the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase and the level of bilirubin (conjugated and unconjugated) differed from the corresponding indexes in the control group (p < 0.05). These change depended on sex. The correlation between the levels of the total bilirubin, activity of the ACT, ALT in serum and absorbed doze to the thyroid of fetus was found (correlation coefficient > -0.8, p < 0.05). The linear regression equations for these three indexes are straight line: the total bilirubin, ACT and ALT decreased with the increase of the absorbed dose to the thyroid of fetus (p < 0.05). The relationship between the all indexes in serum and effective doze and levels of cesium accumulation was not detected.     

A new flow cytometric method to follow DNA gap filling during nucleotide excision repair of UVc-induced damage

BACKGROUND: Several methods have been developed for studying the kinetics of DNA repair after exposure of cells to ultraviolet (UV) light, such as conventional assays measuring unscheduled DNA synthesis (UDS). In this study, we have developed an accurate and rapid method to follow DNA gap filling during nucleotide excision repair (NER) in normal human fibroblasts (NHFs) in response to UV-induced damage. METHODS: After UVc irradiation, aphidicolin was added to the culture to hold repair patches open. This allowed an efficient incorporation of biotin-21-dUTP during an endogenous DNA repair synthesis that was detected by flow cytometry. RESULTS: We showed that the DNA gap filling after UVc irradiation in NHFs increased with time up to 10 h after irradiation and that no repair synthesis activity could be detected in XP-A fibroblasts. Furthermore, this activity was UVc dose dependent up to 20 J/m2. These results correlated well with those of the UDS assay. Interestingly, addition of aphidicolin at different time points after UVc irradiation, thus allowing endogenous repair synthesis in the absence of biotin-21-dUTP, demonstrated that the response of the NER system occurred extremely rapidly after irradiation. CONCLUSIONS: This method may be a reliable and simple alternative to other techniques measuring UDS. Practical advantages include the rapidity of the method, no need for radioactivity, and the possibility to use a second and/even a third flow marker to analyse cell cycle and heterogeneous cell populations concomitantly.     

X-ray dose-effect relationship on unscheduled DNA synthesis and spontaneous unscheduled DNA synthesis in mouse brain cells studied in vivo

Summary X-irradiation of the head of adult mice leads to DNA repair synthesis (unscheduled DNA synthesis, UDS) in non-proliferating cells of the brain as shown autoradiographically after injection of³H-thymidine and subsequent irradiation. The extent of UDS induced by one and the same X-ray dose varies between different cell types and also between different brain areas. Within the range of X-ray doses studied (2 to 100 Gy) a linear dose effect relationship was observed. No evidence of a saturation effect was found. The slopes of the regression lines for the dose effect relationship differ considerably for the different cell types. Two interesting correlations were found, if the present results were compared with other data in the literature: (i) There seems to be a correlation between the extent of UDS and radiosensitivity of the different cell types, the cells with low DNA repair synthetic rates being more radiosensitive. (ii) The extent of UDS of the different cell types correlates well with the extent of protein synthesis of the corresponding cell types. Apart from radiation induced UDS, spontaneous UDS was found to occur in sham-irradiated animals. The extent of spontaneous UDS also differs considerably between different cell types as well as between different brain areas. The increase of spontaneous UDS with increasing duration of immobilization of the animals during sham irradiation suggests a relationship between spontaneous UDS and stress.     

Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.     

Kinetics of mitotic rhythmic activity in oat (Avena sativa) coleoptiles following irradiation

The analysis of different intensity (10-200 Gy) irradiation influence on rhythmical processes in mitotic activity of oats coleoptiles was carries out. Obtained results indicate that irradiation disorders phase and amplitude of the cell division rhythm in investigated material. This disturbance, as judged by doze dependence, is associated with unknown molecular structures.     

Effect of continuous, whole-body gamma irradiation upon canine lymphohematopoietic (CFU-GM, CFU-L) progenitors and a possible hematopoietic regulatory population

Clonogenic assays for granulocytes-macrophages (CFU-GM) in bone marrow and for T lymphocytes (CFU-L) in peripheral blood were performed on dogs continuously exposed to 60Co irradiation (0.02, 0.04, or 0.11 Gy/day). When decreased numbers of CFU-GM were observed they correlated well with the clinical status of the dogs but were not generally associated with increasing cumulative doses of absorbed irradiation. In clinically normal, irradiated animals, decreased CFU-GM values and myeloid-erythroid ratios were observed, suggesting that chronic irradiation may affect the granulocytic series well before decreased peripheral blood values are seen. In hypocellular dogs the number of CFU-GM were significantly decreased compared to values obtained from control or clinically normal irradiated dogs, while virtually no CFU-GM were observed in the leukemic dogs. Only the CFU-GM values of the hypocellular group showed an association, e.g., a suggestion of an abortive regenerative effort, with increasing absorbed dose. Proliferative capacity of T lymphocytes (CFU-L) was not affected by either increasing absorbed irradiation or the presence of leukemia. D0 values were determined on marrow fibroblastic cells to ascertain whether a radioresistant subpopulation of stromal elements would result from continuous in vivo irradiation. No correlation was found between absorbed dose and increased D0 values. However, seven of eight dogs which developed acute nonlymphocytic leukemia displayed marrow fibroblastic cells with elevated D0 values. These radioresistant marrow fibroblastic cells were assayed for their ability to support normal granulopoiesis and found to be not significantly different from control fibroblasts.     

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis

Factory workers exposed to ethylene oxide (EO), 0.5–1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes.The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = ?0.45, p < 0.02) and the number of chromosome breaks (r = ?0.61, p < 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected.It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.     

Influence of in vitro low-level gamma-radiation on the UV-induced DNA repair capacity of human lymphocytes – analysed by unscheduled DNA synthesis (UDS) and comet assay

Unscheduled DNA synthesis (UDS) induced by ultraviolet radiation (UV) was studied in human lymphocytes after exposing blood samples in vitro to doses ranging between 1 and 10 mGy gamma-radiation, by way of measuring tritiated thymidine (³H-TdR) uptake in the DNA of these lymphocytes. The results indicate that samples pre-exposed to gamma-ray doses ranging between 2.5 and 4 mGy show higher UDS levels compared with those pre-exposed to doses of less than 2.5 or more than 4 mGy. These results were verified by studying the rate of removal of UV-induced photoproducts using the comet assay. The reason for the increase in DNA repair capacity in this dose range is discussed in comparison with earlier reports on this phenomenon. The DNA repair capacity with respect to inter-individual variability and age is also analysed. The study implies that the comet assay is a simple and sensitive visual method to track nucleotide excision repair and hence can be used to estimate UV-induced DNA repair in the place of the more reliable yet cumbersome and time-consuming, grain-counting autoradiographic technique. Received: 28 April 1998 / Accepted in revised form: 1 September 1998     

DNA repair synthesis and chromosomal aberrations induced in vivo by triethylenemelamine

The alkylating agent, triethylenemelamine (TEM), was studied for its ability to induce unscheduled DNA (repair) synthesis (UDS) in vivo in rat lymphocytes. Somatic cytogenetic alterations were analyzed (in bone marrow) and compared with UDS as a function of TEM dosage. UDS was evaluated through the use of autoradiography; cytogenetic alterations were studied in metaphase bone marrow chromosome preparations.Data indicated that the degree of UDS is a direct function of TEM dosage up to a rate-limiting concentration, at which point it ceases to be dose dependent. Except for a deviation at the highest dose level tested, the extent of cytogenetic damage was directly and linearly related to TEM dose. Between the control and intermediate (0.2 mg/kg) dose levels, UDS response increased II-fold while cytogenetic damage showed only a 4-fold increase; this disparity diminished with increasing TEM dose. In the lower dose levels, therefore, the greater relative sensitivity of UDS evaluation in the detection of genetic activity may be indicated. Patterns of UDS response observed through the in vivo assay developed in this study were found to be analogous to those established in in vitro studies.     

Ultraviolet light sensitivity,unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of ³H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m²), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.     

Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum

Summary The distribution of spontaneous sister chromatid exchanges (SCEs) was studied in PHA-stimulated lymphocytes from 15 patients affected by xeroderma pigmentosum (XP). The study of unscheduled DNA synthesis (UDS) in twelve of these patients showed that seven were deficient and five proficient. The number of SCEs in XP patient cells was higher than in those of 19 controls, and the distributions of SCEs per cell were significantly different. However, the results varied when XP patients were considered in relation to their UDS: the group of XP patients with proficient UDS did not differ, whereas the group of XP patients with deficient UDS was very significantly different from controls. The group not tested for UDS was similar to the deficient UDS group. The possible relationship between the increase of SCEs and the type of DNA repair defect is discussed.