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1.
Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.  相似文献   

2.
Detection of radiation-induced apoptosis using the comet assay   总被引：2，自引：0，他引：2  
The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis.  相似文献   

3.
Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.  相似文献   

4.
The amounts of DNA single strand breaks that are oxidative damage produced by copper were examined by comet assay in the liver cells of an inbred strain of Long-Evans Cinnamon (LEC) rats that spontaneously develops fulminant hepatitis. At 4 weeks of age, copper contents in the liver of LEC rats were approximately 30-fold higher than those of WKAH rats that are control rats used in the present study. Copper accumulated in the liver of LEC rats in an age-dependent manner and no significant differences were observed between copper contents in the livers of males and females at each week of age from 4 to 15 weeks. No significant amounts of DNA strand breaks were found in the liver cells of both male and female WKAH rats from 4 to 15 weeks of age. DNA strand breaks were produced in the substantial population of LEC rat liver cells at 10 weeks of age and induced in an age-dependent manner from 10 to 15 weeks of age. The amounts of DNA strand breaks produced by copper accumulation in the liver cells of female LEC rats are not more abundant than those in the cells of male rats, although it has been reported that hepatitis in female rats is more serious than that in male rats.  相似文献   

5.
利用碱性单细胞凝胶电泳技术研究洛克沙胂对中国仓鼠肺细胞(CHL)的DNA损伤影响。洛克沙胂分为10、100、500、1000mg/L剂量组,分别以磷酸盐缓冲液和10mg/L亚砷酸钠为对照组,经3、6、12、24、48h暴露后进行单细胞凝胶电泳。结果表明,慧星试验参数尾DNA含量、慧星全长、慧尾长、尾距和Olive尾距等表现出剂量-效应、时间-效应关系,不同剂量洛克沙胂、不同暴露时间下对CHL细胞有不同程度的DNA损伤。  相似文献   

6.
Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/β ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/β ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.  相似文献   

7.
试验旨在通过改良彗星实验检测黄曲霉毒素B1（aflatoxin-B1,AFB1）对雏鸭肝细胞DNA损伤的影响。雏鸭经AFB1灌胃染毒,2 h后分离肝细胞,并通过改良彗星实验测定DNA损伤。结果显示,AFB1能够导致雏鸭肝细胞DNA损伤,表现为〖JP2〗尾长、尾部DNA百分含量、尾矩、Olive尾矩等彗星参数与空白和溶剂对照组相比显著增加（P<0.05）。表明改良彗星实验能够用于AFB1导致肝细胞DNA损伤的检测,试验还提示,在体肝细胞彗星实验能够作为雏鸭AFB1暴露的遗传毒性标志物。  相似文献   

8.
Carbon tetrachloride (CCl(4)) -induced hepatotoxicity is a commonly used model for investigating lipid peroxidation-related tissue injury. In the present study, the effect of flaxseed extract was observed on histological sections, glutathione-content and DNA strand breaks. Lignan-containing flaxseed extract (1.6 g/kg body weight/day) was daily administered with intragastric injection to rats for three days, on the fourth day, CCl(4) (2 g/kg) was intraperitoneally injected. Liver tissue was sampled at 24 hr after administering CCl(4). Liver-necrosis was observed in CCl(4)-injected rats without pretreatment of flaxseed extract. Pretreatment of flaxseed extract reduced extent of the necrosis found 24 hr after the intraperitoneal administration of CCl(4). Pretreatment of flaxseed extract protect against CCl(4)-induced decrease of reduced glutathione-content measured from reactions with 5,5'-dithiobis-(2-nitrobenzoic acid) and also protect against the elevation of DNA strand breaks in the liver cells measured by comet assay. Flaxseed-extract appears to protect liver cells against CCl(4)-induced necrosis.  相似文献   

9.
改良彗星实验检测黄曲霉毒素B_1致雏鸭DNA损伤   总被引：1，自引：0，他引：1  
试验旨在通过改良彗星实验检测黄曲霉毒素B1(aflatoxi-B1,AFB1)对雏鸭肝细胞DNA损伤的影响.锥鸭经AFB1灌胃染毒,2 h后分离肝细胞,并通过改良彗星实验测定DNA损伤.结果显示,AFB1能够导致雏鸭肝细胞DNA损伤,表现为尾长、尾部DNA百分含量、尾矩、Olive尾矩等彗星参数与空白和溶剂对照组相比显著增加(P<0.05).表明改良彗星实验能够用于AFB1导致肝细胞DNA损伤的检测,试验还提示,在体肝细胞彗星实验能够作为雏鸭AFB1暴露的遗传毒性标志物.  相似文献   

10.
Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.  相似文献   

11.
Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metastatic tumour growth and may influence proliferation, survival, metastasis and angiogenesis. AR antagonists like propranolol and carvedilol inhibit proliferation, induce apoptosis and synergize with chemotherapy agents in some cancers. Radiation resistance is mediated in many cells by upregulation of pro‐survival pathways, which may be influenced by ARs. Studies evaluating AR antagonists combined with radiation are limited. The purpose of this study was to determine the effect of propranolol and carvedilol on viability and radiosensitivity in sarcoma cell lines. The hypothesis was that propranolol and carvedilol would increase radiosensitivity in four primary bone sarcoma cell lines. Single agent propranolol or carvedilol inhibited cell viability in all cell lines in a concentration‐dependent manner. The mean inhibitory concentrations (IC50) for carvedilol were approximately 4‐fold lower than propranolol and may be clinically relevant in vivo. Immunoblot analysis confirmed AR expression in both human and canine sarcoma cell lines; however, there was no correlation between baseline AR protein expression and radiosensitivity. Short duration treatment with carvedilol and propranolol did not significantly affect clonogenic survival. Prolonged exposure to propranolol and carvedilol significantly decreased the surviving fraction of canine osteosarcoma cells after 3Gy radiation. Based on our results and possible in vivo activity in dogs, further studies investigating the effects of carvedilol on sarcoma are warranted.  相似文献   

12.
A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.  相似文献   

13.
Diagnosis of the fertilizing ability of a semen sample is important for consistently high reproductive efficiency. Disturbances in the organization of the genomic material in sperm nuclei can have a serious impact on the growth of the offspring, therefore a stable nuclear matrix is crucial for participation in embryonic development. Routine semen analysis investigates parameters such as sperm motility and morphology, but does not examine the nuclear DNA integrity of spermatozoa. It has been suggested that altered nuclear chromatin structure or damaged DNA in spermatozoa is implicated as a possible cause of increased infertility in males. Therefore, it is crucial to develop and use accurate and diagnostic tests, which may provide better prognostic capabilities than the standard sperm assessments. This article reviews and discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. These different techniques include the comet assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), tritium-labelled 3H-actinomycin D (3H-AMD) incorporation assay, terminal TdT-mediated dUTP-nick-end labelling (TUNEL) assay, in-situ nick translation (ISNT) assay, DNA breakage detection-fluorescence in-situ hybridizations (DBD-FISH) assay and sperm chromatin dispersion (SCD) test. The aforementioned assays, which are considered independent measure of sperm quality, may help to detect subtle defects in the chromatin structure or DNA integrity, and thereby assist in semen quality assessment. The relationship between DNA damage and male infertility is also addressed.  相似文献   

14.
A normal prion protein (PrPc) is converted to a protease-resistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.  相似文献   

15.
Considering the high incidence of dogs with acute bacterial cystitis (BC) and the relationship among inflammation, genotoxicity, and carcinogenesis, we conducted a case-control study comparing the frequency of deoxyribonucleic acid (DNA) lesions assessed by the comet assay between disease-free animals (13 males and 13 females) and cytology-confirmed cases of acute BC (12 males and 12 females), which was mainly caused by Staphylococcus sp. (40%) and Escherichia coli (35%). The results show no increase in DNA damage in cells obtained by bladder washings and no influence of age, sex, and breed due to acute BC. In conclusion, DNA damage was seemingly not associated with the infection by specific bacteria.  相似文献   

16.
The consumption of fruits and vegetables is associated with a reduced risk of various ailments, including cancer and cardiovascular diseases. Carotenoids, such as lycopene and beta-carotene, are natural constituents of edible plants and may protect against disease. In this study, the influence of lycopene and beta-carotene on DNA damage caused by catechol-estrogens in vitro is examined. One possible mechanism by which catechol estrogens such as 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol, which cause DNA damage in naked plasmid DNA as well as in cells, contributing to the process of carcinogenesis, is through the generation of reactive oxygen species. It was found that both carotenoids at concentrations ranging from 0.25 to 10 microM significantly inhibit strand breakage induced by 4-OHE2/copper sulphate by up to approximately 90% in plasmid DNA with beta-carotene being slightly more effective. No prooxidant or cytotoxic effects were observed at the concentrations tested. These carotenoids had a similar, though reduced effect on DNA damage as measured by the comet assay, in Chinese hamster lung fibroblasts. The results obtained show that both lycopene and beta-carotene, most probably and mainly through their potent antioxidant properties, are able to inhibit catechol-estrogen-mediated DNA damage.  相似文献   

17.
Rapamycin has been reported to reduce cancer cell survival in certain tumors following radiation therapy, but the mechanisms driving this phenomenon are unclear. Rapamycin inhibits mTOR signaling, a pathway responsible for several essential cell functions. The objective of this study was to investigate the effects of rapamycin and radiation on the activation and inhibition of mTOR signaling and the relationship between mTOR signaling and DNA damage response in vitro using canine mast cell tumor (MCT) cancer cell lines. Rapamycin rapidly inhibited S6K phosphorylation in a dose-dependent manner. Ionizing radiation (3, 6, or 10 Gy) was able to activate mTOR signalling, but the combination of radiation and rapamycin maintained mTOR inhibition. The comet assay revealed that co-treatment with rapamycin induced modest increases in the severity of DNA damage to MCT cells, but that these differences were not statistically significant. Although the relationship between mTOR and DNA damage response in MCT cancer cell lines remains unclear, our findings suggest the possibility of interaction, leading to enhancement of radiation response.  相似文献   

18.
本文为确定性控冻精与常规冻精的DNA损伤程度,利用彗星实验法分别将奶牛的性控冻精和常规冻精凝胶混合液在裂解液中作用后,使用普通载玻片双层铺胶,电泳,吖啶橙染色后在荧光显微镜下检测、拍照,经CASP软件图像分析后用SPSS 11.5进行数据统计,初步建立了奶牛精子DNA损伤的5级彗星图谱。统计结果显示,性控冻精的DNA损伤系数为116,显著高于常规冻精的损伤系数（28）（P〈0.01）;彗星率为63.06%,显著高于常规冻精的彗星率（31.13%）（P〈0.01）。可以得出,性控冻精DNA损伤显著高于常规冻精。  相似文献   

19.
The effects of dietary nucleotides on thymocyte DNA damages induced by cyclophosphamide (CP) in mice were examined. First, phase I experiment was conducted to determine the optimal timing of detecting thymocyte DNA damages induced by CP (150 mg/kg body weight) in mice. Thymocyte DNA damages was determined at 6, 12, 18, 24 h by single-cell gel electrophosphoresis assay (comet assay) after intraperitoneal injection of CP. The levels of DNA damage at 6, 12, 18, 24 h were all significantly higher than that of the control group (p < 0.01). The highest level of DNA damage appeared at 18 h and then decreased at 24 h. Therefore, 18 h was selected to determine DNA damages induced by CP in subsequent experiments. In phase II experiment, 30 male KunMing mice were divided into three treatments: negative control (NC), positive control (PC) and nucleotides group (NG). Mice in NC and PC were fed nucleotide-free diet, and mice in NG were fed nucleotide-supplemented diet (supplemented with 0.25% nucleotides, a mixture containing equal amounts of AMP, CMP, GMP and UMP). Mice in PC and NG groups were injected with CP (150 mg/kg body weight) at 21 days. DNA damage in thymocytes was evaluated at 18 h after CP treatment. The results indicate that dietary nucleotides do not affect the weights of the thymus and the spleen, or their organ indices (p > 0.05), but significantly decrease the percentage of comet cells and comet tail sizes (p < 0.01). This study demonstrates that dietary nucleotides could reduce the level of thymocyte DNA damage induced by CP in mice.  相似文献   

20.
This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.  相似文献   

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