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OBJECTIVE: To investigate the sensitive target cell population of spermatocyte injury induced by TL in mice. METHODS: Seven to eight-week-old healthy male C57BL/6 mice were orally administered triptolid(TL) of 0.125, 0.25 and 0.5 mg•kg-1 per day, and dissected on days 3, 7, 11 and 15 respectively. The extracted testes were fixed with formaldehyde. Paraffin sections and hematoxylin-eosin (HE) staining were performed to determine the optimal time point and dose level to be applied for sensitive target cell population analysis of spermatocyte injury induced by TL. The primary spermatocytes in different stages were clearly distinguished and counted based on the characteristic distribution profile of γ-H2AX in spermatocytes under immunohistochemical staining. The sensitive target cell populations of spermatocyte injury were determined according to the decreased percentage of spermatocytes in different stages. RESULTS: HE staining showed that the best dose-effect relationship of spermatogenic cell injury in the testes was present on day 11 after TL administration (The severity of the lesion ranged from a minimal degree in the 0.125 mg•kg-1 group to a mild to moderate injury in the 0.25 mg•kg-1 group, and finally to a marked injury in the 0.5 mg•kg-1 group). The degree of injury in the 0.125 and 0.25 mg•kg-1 groups was appropriate and suitable for determination of sensitive target cell populations. γ-H2AX immunohistochemical staining indicated that the γ-H2AX showed different distribution characteristics in nucleus in different stages of spermatocyte differentiation: scattered throughout the nucleus in a few discrete foci to fill the whole nuclear in leptotene; assembled in the chromatin regions in zygotene; located on the edge of the nucleus in a single foci in the pachytene; located in the nucleus in a single foci in the diplotene. The counting results showed that the absolute number of primary spermatocytes in all differentiating stages decreased slightly without statistical significance (P>0.05) in the 0.125 mg•kg-1 dose group; the absolute number of primary spermatocytes decreased significantly with statistical significance (P<0.01 or 0.001) in the 0.25 mg•kg-1 dose group. There was higher decreased percentage of the leptotene primary spermatocyte among the differentiating stages before pachytene stage and with statistical significance (P<0.05) at 0.25 mg•kg-1 when compared with the pachytene primary spermatocyte. CONCLUSION: γ-H2AX immunohistochemical staining can clearly distinguish primary spermatocytes at different stages. The leptotene primary spermatocytes are the most likely sensitive target cells in the testicular spermatocyte-injury induced by TL administration.
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Aims:Non-small cell lung cancer (NSCLC) accounts for high lung cancer death that is mostly associated with advanced disease stage at diagnosis and resistance to chemotherapy. In the present study, we investigated whether xanthohumol, a prenylated flavonoid of hop plant, induces metastatic lung cancer H1299 cell death, and whether in combination with cisplatin there are additive effects. Methodology:H1299 cells were grown and treated with xanthohumol (6.25, 12.5, or 25 μM), cisplatin (12.5, 25, or 50 μM) and the combination of cisplatin and xanthohumol for 24 h. Cell viability, cell morphology, chromatin condensation, ɣH2AX, cPARP-1, capsase-3, p21WAF1/CIP1and p14ARFgenes were analyzed Results:Xanthohumol, cisplatin, and the combination of cisplatin and xanthohumol inhibited H1299 cells viability. Cisplatin growth inhibitory effects were potentiated by xanthohumol. Xanthohumol induced chromatin condensation and apoptosis and potentiated cisplatin’s effect vs cisplatin alone. Further investigation of growth inhibitory effects, xanthohumol alone induced γH2AX foci formation and the combination potentiated γH2AX foci formation. Cisplatin, xanthohumol at 25 μM, and the combination of cisplatin and xanthohumol at 6.25 and 12.5 μM increased cPARP-1 level. Active caspase-3 was increased by cisplatin, 12.5 μM of xanthohumol, and the combination of xanthohumol and cisplatin. Xanthohumol at 6.25 or 12.5 μM potentiated cisplatin effect on active caspase-3 and cPARP-1, respectively. Xanthohumol at 25 μM significtly induced the expression cell cycle control genes p21WAF1/CIP1and p14ARF. These results indicate that xanthohumol inhibits proliferation of H1299 cells and induces cell death through cleavage of PARP-1 and activation of caspase-3. The combination of cisplatin and xanthohumol potentiated cytotoxic effects of each other compound.Conclusion:The present study suggests that xanthohumol poses apoptotic effects and potentiates cisplatin’s growth inhibitory effects on metastatic lung cancer cells
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Objective To investigate the expression of bone marrow γ-H2AX in the patients with multiple myeloma(MM) and its correlation with the prognosis.Methods The patients with newly diagnosed MM in this hospital were selected as the case group,and the patients with non-hemopoietic system tumor without obvious morphological abnormalities by bone marrow smear and biopsy served as the control group.The immunohistochemistry was adopted to detect the expression level of bone marrow γ-H2AX in the cases group and control group,the image-Pro Plus(IPP) semiquantitative analysis was performed.The expression differences were compared between the two groups,moreover the case group was re-divided into the strong expression group and weak expression group according to γ-H2AX expression level.Then the relation ship between γ-H2AX expression level and the prognosis in the patients with MM.Results The bone marrow γ-H2AX expression level in the case group was significantly higher than that in the control group (P＜0.05);the level of γ-H2AX expression in the strong expression group was significantly stronger than that in the weak expression group (P＜0.05).Conclusion The level of γ-H2AX expression was higher among MM patients,and the over expression of γ-H2AX predicts the shorter survival time.
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AIM To investigate the effects of Wuzi Yanzong Prescription (Lycii Fructus,Cuscutae Semen,Rubi Fructus,Plantaginis Semen and Schisandrae chinensis Fructus) on DNA damage of testis cells in adult male mice induced by cyclophosphamide (CTX).METHODS Forty out of fifty adult male Balb/C mice were injected intraperitoneally with CTX and then were randomly and equally divided into model control group (normal saline),Wuzi Yanzong Prescription low-,medium-and high-dose groups (100,200 and 400 mg/kg),and the other ten mice served as normal control group (normal saline).All mice were anesthetized by inhalation of ether,and then were sacrificed by cervical dislocation.The sperm count,sperm motility and malformation rate of sperm were tested.The content of 8-hydroxy-deoxyguanosine (8-OHdG) in serum was measured using ELISA;the DNA damage degree of cells in testis was detected by single cell gel electrophoresis;the protein expressions of p-P53 and γ-H2AX in testis were examined by Western blot.RESULTS Compared with the model control group,Wuzi Yanzong Prescription groups showed significant increase in the sperm count,sperm motility and significantly decreased malformation rate of sperm,the level of 8-OHdG in serum,and the protein expressions of p-P53 and γ-H2AX in testis were also significantly decreased.CONCLUSION Wuzi Yanzong Prescription can significantly alleviate the DNA damage of testis cells in mice induced by CTX through down-regulating protein expressions of p-P53 and γ-H2AX.
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Objective To evaluate the effect of iodine contrast agent on the biological responses of CT examination. Methods A total of sixty patients with suspected urinary tract disease who underwent computed tomography urography ( CTU ) examination were randomly divided into control group and experimental group. The control group was treated with routine CTU, where only CT scan was performed on the first day. CTU was added after 3 days. The test group was treated with fractional injection CTU and injected with enhanced scanning agent on the first day. Before and after CT examination, the patients′peripheral blood was collected and the number of γ-H2AX foci in lymphocytes ( mononuclear cells) was measured by immunofluorescence, and the differences of DNA damage in these two groups were observed. Results Before and after CT examination, the number ofγ-H2AX foci was 0. 06 ± 0. 02 and 1. 06 ± 0. 27 in the lymphocytes of control group,0. 06 ± 0. 03 and 1. 42 ± 0. 50 in the test group, respectively. Hence, the number ofγ-H2AX foci in the test group was increased by 38. 14%. Moreover, the change ofγ-H2AX foci in these two groups was not influenced by gender, but correlated with ages( between≤50 years old and>50 years old) in control group (t= -4. 76, P<0. 05) and in test group(t= -8. 16, P <0. 05). Conclusions The iodine contrast agent can increase DNA damage of CT examination, and therefore the use of iodine contrast agent in CT should be reduced as much as possible in clinical work.
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Radiation biodosimetry is an important part of radiation medicine. Some new discoveries and progresses have been made in radiation biodosimetry studies in recent years. Cytogenetic method represented by chromosome aberration analysis as the golden standard of radiation biodosimeter is being transformed to automate analysis, and a number of international, regional and national laboratory networks of radiation biodosimetry are being established. As a widely acceptable molecular marker of DNA damage,γ-H2AX has made rapid progress in radiation dose estimation. Based on the expressions of protein and genes, further advancements have been made in the studies of metabolites and miRNAs. At the same time, with the development of proteomics technology, there are some breakthroughs in the study of using molecular expression profiling to evaluate radiation dose. The research progresses of radiation biodosimetry is reviewed in this paper.
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Radiation biodosimetry is an important part of radiation medicine. Some new discoveries and progresses have been made in radiation biodosimetry studies in recent years. Cytogenetic method represented by chromosome aberration analysis as the golden standard of radiation biodosimeter is being transformed to automate analysis, and a number of international, regional and national laboratory networks of radiation biodosimetry are being established. As a widely acceptable molecular marker of DNA damage,γ-H2AX has made rapid progress in radiation dose estimation. Based on the expressions of protein and genes, further advancements have been made in the studies of metabolites and miRNAs. At the same time, with the development of proteomics technology, there are some breakthroughs in the study of using molecular expression profiling to evaluate radiation dose. The research progresses of radiation biodosimetry is reviewed in this paper.
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Objective To synthesize novel gold nanoparticles of GAL-PEG-GNPs,study its radiation effect on hepatocellular carcinoma cells HepG2 cells in vitro,and investigate the underlying mechanisms.Methods GAL-PEG-GNPs were synthesized and characterized successfully.HepG2 cells were divided into three groups of control,GNPs and GAL-PEG-GNPs.The cytotoxicities of these compounds were tested by the CCK-8 assay and their IC50 values of HepG2 cells were calculated.Cell uptake of nanoparticles was detected by TEM and ICP-MS.The radiosensitization effect of nanoparticles was tested by the colony formation assay.Cell cycle distribution was detected by FCM.The expressions of CAT,SOD,and total GSH were detected with a microplate reader,and the expressions of apoptosis-related proteins were tested by Western blot.Results The GNPs and GAL-PEG-GNPs had absorption peaks at 520 and 530 nm,respectively,and their diameters were (22.6-±2.12) and (32.0 ± 1.41) nm detected by ICP-MS.The GAL-PEG-GNPs and GNPs had similar cytotoxicity profiles (P ＞ 0.05),while GAL-PEG-GNPs could be more effectively uptaken by HepG2 cells than GNPs.The sensitive enhancement ratio (SER) of GNPs and GAL-PEG-GNPs to HepG2 cells were 1.46 和 1.95,respectively.The percentage of cells at phase of G2/M in HepG2 population treated with GNP was higher than that of untreated cells (t =14.20,P ＜0.05).The protein expressions of Cytochrome C,Bax,Caspase-3,and Caspase-9 were upregulated while Bcl-2 expression was down-regulated in the cells treated with GNPs/radiation or GAL-PEGGNPs/radiation.The expressions of CAT,SOD and total GSH in the GNP treated groups were significantly decreased compared with the control group(t =12.34,29.39,12.85,P ＜ 0.05).Conclusions GALPEG-GNPs has obvious radiosensitization effect on HepG2 cells,which is related to the induction of cell apoptosis and the generation of free radicals.
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Objective To investigate the influence of MgSO4 with different concentrations on cell survival and γ-H2AX expression in HUVEC irradiated with X-rays.Methods Cell proliferation rate was assayed by CCK-8,γ-H2AX foci formationwas observed with a laser confocal microscope,and γ-H2AX protein expression was detected by flow cytometer and Western blot assay.Results MgSO4 with a concentration of 1.25 mg/ml could improve the survival rate of IHUVEC treated with X-rays (t =-6.34,P ＜ 0.05).After irradiation,γ-H2AX loci approached to the highest level from 30 min to 1 h after radiation and then decreased.MgSO4 significantly reduced the foci formation at 0.5,1,2,6 and 12 h postirradiation (t =12.62,6.36,11.93,5.75,9.43,P ＜ 0.05).Both flow cytometry,and Western blot assays showed that MgSO4 inhibited γ-H2AX protein expression at 0.5,1 and 2 h post-irradiation (t =6.07,5.32,11.85,P ＜ 0.05).Conclusions MgSO4 could improve the survival rate and reduced γ-H2AX expression in HUVEC irradiated with X-ray.
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Objective To evaluate the potential feasibility of γ-H2AX foci as a biodosimetry after exposure to ionizing radiation by comparing DNA double-strand break repair kinetics in rat blood lymphocytes with that in human lymphocytes.Methods Peripheral blood lymphocytes separated from Sprague-Dawley(SD) male rats and healthy adults were exposed to γ-rays,and some rats were also subjected to total body irradiation.The inductions of DNA repair-related foci of γ-H2AX,pATM (S1981) and pDNA-PKcs (T2609) were detected with immunofluorescence staining technique at different time points post-irradiation,and the status of their co-localization was analyzed.Results The induction kinetics of γ-H2AX foci in rat lymphocytes was similar to that observed in human lymphocytes.The frequencies of γ-H2AX foci peaked at 30 min after γ-ray irradiation (trst =62.64,th =28.52,P ＜ 0.05),then decreased rapidly after 6 h post-irradiation (trat =45.96,th =14.80,P ＜0.05),and the residual foci number remained only about 3％-8％ of its maximal value at 24 h post-irradiation.At 30 min after γ-ray irradiation,the frequencies of pATM (S1981) and pDNA-PKcs (T2609) foci in rat and human lymphocytes significantly higher than those of nonirradiated control (trat =21.05,25.80,th =11.07,29.52,P ＜ 0.05),and the frequencies of co-localization of pATM (S1981) or pDNA-PKcs (T2609) foci with γ-H2AX foci also markedly increased by 26％-32％ in irradiated lymphocytes of rat and human (trat =5.34,9.14,thuman =18.32,51.28,P ＜0.05).Moreover,γ-H2AX foci incidence in rat lymphocytes in vitro was consistent with that induced by total body irradiation of rat.The number of γ-H2AX foci in irradiated rat lymphocytes increased with irradiation dose in a linear dose-dependent manner,its slope was similar to that of irradiated human lymphocytes reported by other laboratory.Conclusions Rat is a useful animal model to evaluate radiation biodosimetry with γ-H2AX foci in lymphocytes.The co-activation of ATM and DNA-PK plays an important role in DSB repair in the irradiated lymphocytes of rat and human.
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AIM:To investigate the effect of MK-2206, an inhibitor of protein kinase B (Akt), on the DNA damage of SGC-7901 cells.METHODS: SGC-7901 cells were treated with different concentrations of MK-2206, and phosphorylated histone H2AX (γ-H2AX) foci formation was detected by immunofluorescence staining .Western blot analy-sis was used to exam the levels of DNA damage-related protein.The expression of LC3-Ⅱ was determined to evaluate the change of autophagy .RESULTS:MK-2206 treatment increased the formation of γ-H2AX foci and histone H2AX phospho-rylation in the SGC-7901 cells.The levels of DNA damage response protein were also increased .In addition, MK-2206-treated SGC-7901 cells increased the expression of LC 3-II, a hallmark of autophagy .Inhibition of autophagy significantly enhanced MK-2206-mediated histone H2AX phosphorylation.CONCLUSION:MK-2206 induces DNA damage and auto-phagy in SGC-7901 cells.Blocking autophagy potentiates the response of MK-2206-induced DNA damage .
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Aim To investigate the effect of BMS- 345541 on the repair of DNA DSBs induced by VP-16 in AML cells and its possible mechanism. Methods The effects of BMS-345541 on the sensitivity of AML cells to VP-16 were determined by MTT. Flow cytome-try ( FCM) was applied to test the level of DNA dam-age, cell cycle progression and apoptosis in AML cells. High content analysis ( HCA) was used to verify the amount ofγ-H2AX,p-ATM,RAD51 in AML cells. Results BMS-345541 could significantly inhibit the proliferation of AML cells induced by VP-16 . BMS- ＜br> 345541 increased the amount of RAD51 foci and p-ATM foci in AML cells treated with VP-16 after 6 hours , which led to increased numbers of cells in the G2/M phases of the cell cycle,then induced apoptotic cell death. Conclusion BMS-345541 sensitizes AML cells to VP-16 via selective inhibition of homologous recombinational repair of DNA double-strand breaks.
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Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.
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The present study investigated the enhanced radiosensitivity of U-251 cells induced by sodium butyrate (NaB) and its possible mechanisms.Increased radiosensitivity of U251 cells was examined by clonogenic cell survival assays.The expression of Ku70 mRNA and protein was detected by using RT-PCR and Western blotting respectively.γ-H2AX foci were measured at different time points after ionizing irradiation alone or combined with NaB treatment.The results showed that cell survival rate was significantly reduced,both D0 and Dq values were decreased (D0:1.43 Gy vs.1.76 Gy; Dq:1.22 Gy vs.2.05 Gy) after the combined treatment as compared with irradiation alone,and sensitivity enhancing ratio (SER) reached 1.23.The average number ofγ-H2AX foci per cell receiving the combined treatment was significantly increased at different time points,and the expression levels of Ku70mRNA and protein were suppressed by NaB in a dose-dependent manner.It was concluded that enhanced radiosensitivity induced by NaB involves an inhibited expression of Ku70 and an increase in γ-H2AX foci,which suggests decreased ability in DSB repair.
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Objective To investigate ff the γ-H12AX foci is a precise index for the DSB formation and repair in mature neurons of brain in vivo after clinically relevant doses irradiation. Methods For the DSB formation experiment, the mature neurons in the neocortex of brain tissue of C57BL/6 mice were analyzed at 10 rain after whole-body irradiation with 0.1, 0.5 and 1.0 Gy. For the DSB repair kinetics experiment, the mature neurons in the neocortex of brain tissue of repair-proficient (C57BL/6 mice) and repair-deficient mouse strains (BALB/c, A-T and SCID mice) were analyzed at 0.5, 2.5, 5, 24 and 48 h after whole-body irradiation with 2 Gy. The mature neurons in the neocortex of brain tissue of sham-irradiated mice of each strain served as controls. γ-H2AX immunohistochemistry and γ-H2AX and NeuN double immunofluorescence analysis was used to measure DSBs formation and repair in the mature neurons in the neocortex of brain tissue of the different mouse strains. Results For the DSB formation experiment, γ-H2AX foci levels with a clear linear dose correlation and very low backgrounds in the nuclei in the neocortex of brain tissue were observed. Scoring the loss of γ-H12AX foci allowed us to verify the different, genetically determined DSB repair deficiencies, including the minor impairment of BALB/c mice. Repair-proficient C57BL/6 mice exhibited the fastest decrease in foei number with time, and displayed low levels of residual damage at 24 h and 48 h post-irradiation. In contrast, SCID mice showed highly increased γ-H2AX foci levels at all repair times (0.5 h to 48 h) while A-T mice exhibited a lesser defect which was most significant at later repair times (≥ 5 h). Radiosensitive BALB/c mice exhibited slighdy elevated foei numbers compared with C57BI./6 mice at 5 h and 24 h but not at 48 h post-irradiation. Conclusion Quantifying the γ-H2AX loci in normal tissue represents a sensitive tool for the detection of induction and repair of radiation-induced DSBs at clinically relevant doses in vivo.