Vanadyl sulfate can differentially damage DNA in human lymphocytes and HeLa cells

Using the comet assay, we showed that vanadyl sulfate induced DNA damage in human normal lymphocytes and in HeLa cells. Vanadyl at 0.5 and 1 mM produced DNA single- and double-strand breaks (SSBs and DSBs) in lymphocytes, whereas in HeLa cells we observed only SSBs. Post-treatment of vanadyl-damaged DNA from lymphocytes with formamidopyrimidine-DNA glycosylase (Fpg), an enzyme recognizing oxidized purines, gave rise to a significant increase in the extent of DNA damage. A similar effect was observed in HeLa cells, but, using endonuclease III, we also detected oxidized pyrimidines in DNA of these cells. There were no differences in the extent of DNA damage in the lymphocytes and HeLa cells in the pH >13 and pH 12.1 conditions of the comet assay, which indicates that strand breaks, and not alkali-labile sites, contributed to the measured DNA damage. Study of DNA repair, determined in the comet assay as an ability of cells to decrease of DNA damage, revealed that HeLa cells retained the ability to repair vanadyl-damaged DNA induced at a ten-fold higher concentration than that in lymphocytes. Incubation of the cells with nitrone spin traps DMPO, POBN and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by vanadyl sulfate. The presence of vitamins A, C or E caused an increase of DNA damage in HeLa cells whereas in lymphocytes such an increase was observed only for vitamin C. Our data indicate that vanadyl sulfate can be genotoxic for normal and cancer cells. It seems to have a higher genotoxic potential for cancer cells than for normal lymphocytes. Vitamins A, C and E can increase this potential.     

TPPS4的电化学分析法，TPPS4和BSA的相互作用以及环糊精对二者作用体系影响的荧光法研究

目的确立一种快速、准确检测水溶性卟啉(TPPS₄)的分析方法,从而进一步了解水溶性卟啉和牛血清白蛋白(BSA)之间相互作用的机制。方法采用电化学法对TPPS₄的极谱伏安行为进行了研究,同时采用电化学法、荧光法和紫外法对TPPS₄与BSA之间的相互作用分别进行了研究。3种方法相互辅证使得试验结果更加可靠。结果在底液NaH₂PO₄-Na₂HPO₄缓冲液(pH 7.18)中,TPPS₄在-0.70 V(vs SCE)处有一个稳定而灵敏的还原峰,其峰电流与TPPS₄浓度在1.0×10^-7～1.0×10^-5 mol·L^-1有良好的线形关系(r²=0.998 3,0.999 3),检测限LOD为3.0×10^-8 mol·L^-1。平均标准回收率为99.59%,精密度较好,RSD为0.56%(n=5)。在NH₄Cl-NH₃·H₂O缓冲液(pH 9.05)中,实验结果表明BSA与TPPS₄相互作用生成1∶1的TPPS₄-BSA超分子体系。另外,加入环糊精体系后,磺丁醚-β-CD(SBE-β-CD)和羟丙基-β-CD(HP-β-CD)均能促进TPPS₄与BSA发生反应。结论建立了一种简单、快速、准确的水溶性卟啉四-(4-磺基苯)卟啉(TPPS₄)的电化学分析方法。卟啉类药物被环糊精包合后更容易与人体内的蛋白质进行作用,环糊精在卟啉类药物的控制、释放中具有重要的作用和意义。     

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by four rat thyroid carcinogens

Four chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measures by the Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the four test compounds: 2,4-diaminoanisole (DAA) from 0.10 to 1.0 mM, 4,4'-methylene-bis(N,N-dimethyl)benzenamine (MDB) from 0.32 to 1.8 mM, propylthiouracil (PTU) from 1.8 to 5.6 mM, and 4,4'-thiodianiline (THA) from 0.032 to 0.18 mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in thyreocytes exposed to DAA but absent after treatment with MDB, PTU, and THA. Consistent with their thyroid-specific carcinogenic activity, all the four chemicals, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid, whereas any substantial evidence of DNA lesions was absent in liver, kidney, and lung, which, with the exception of liver tumors caused by THA, are not targets of the carcinogenic activity of the four test compounds. These findings indicate that the DNA damage observed in thyroid cells was consistent with the carcinogenicity of the four test compounds, and suggest that DAA, MDB, PTU, and THA might be carcinogenic to thyroid in humans.     

姜黄素对子宫颈癌HeLa细胞的抑制作用

目的：探讨姜黄素对子宫颈癌HeLa细胞的抑制作用及其机制。方法：以细胞培养，镜下观察细胞形态学改变和计数法测定生长曲线；用^3H-脱氧胸苷掺入法测定对DNA合成的影响，以MTT比色法检测给药后HeLa细胞的增殖抑制情况。结果：姜黄素作用HeLa细胞后，癌细胞生长延缓并萎缩，胞质粗糙，有大量颗粒状物堆积，而且药物浓度越大，形态学改变越明显；生长曲线测定、^3H-脱氧胸苷掺入法及MTT比色法实验结果显示，姜黄素对HeLa细胞的增殖和生长有显著的抑制作用并呈明显的时间一剂量依赖关系，当姜黄素浓度为20μg／ml以上时，其对HeLa细胞的掺入抑制率高于5-Fu 20μg/ml对该细胞的掺入抑制率。结论：姜黄素对HeLa细胞具有直接杀伤作用，其机制可能通过干扰细胞代谢，改变细胞外膜的性质抑制肿瘤细胞增殖。     

Genotoxicity of sodium arsenite and DNA fragmentation in ovarian cells of rat

Present study examined the genotoxic effects of arsenite in ovarian tissue of rat at 56 days of age. Immature (28 days old) female rats were exposed to different doses (50, 100, and 200 ppm) of sodium arsenite in drinking water for 28 days. DNA damage in ovarian tissue was measured by comet assay. All doses induced significant decrease in ovarian weight in a dose-dependent manner compared to control, more prominently at (P < 0.001) 100 and 200 ppm. All the comet assay parameters showed significant difference with arsenite treatment compared to control group. In treatment groups, mean number of cells with intact DNA decreased while, mean comet number increased (P < 0.001) in a dose-dependent manner compared to control. Significant decrease (P < 0.05) was observed in mean comet length, height, comet head diameter and %DNA in comet head of high dose groups compared to control group. Dose dependent increase was found in mean comet tail length, %DNA in tail, tail moment and olive tail moment in high dose groups compared to control group. The study indicates that arsenic caused DNA damage to ovarian cells particularly at high doses and ensure comet assay as an effective method to detect DNA damage in tissue caused by metals.     

The effect of quercetin on pro-apoptotic activity of cisplatin in HeLa cells

It is well known that some tumour cells are very resistant to chemotherapy-induced cell death which indicate poor prognosis for patients. Thus the aim of the present study was to investigate the effect of quercetin on pro-apoptotic activity of cisplatin in human cervix carcinoma cells (HeLa). Three variants of experiments were performed. In the first one cells were incubated with studied drugs separately for 8 and 24h. In the second, drugs were added to the culture medium simultaneously. In third cisplatin or quercetin addition was followed by subsequent quercetin or cisplatin treatment, respectively. We observed different apoptotic effects, dependent on the drug succession. Preincubation of cells with quercetin followed by cisplatin treatment appeared to be the most effective and was correlated with strong activation of caspase-3 and inhibition of both heat shock proteins (Hsp72) and multi-drug resistance proteins (MRP) levels. Our results indicate that quercetin pretreatment sensitizes HeLa cells to cisplatin-induced apoptosis in HeLa cells.     

姜黄素对长波紫外线所致DNA损伤的拮抗作用

目的　观察长波紫外线引起人表皮样癌细胞 DNA链断裂损伤的情况 ,以及姜黄素对这种损伤的拮抗作用。方法　用长波紫外线照射细胞 ,用单细胞凝胶电泳法检测 DNA损伤。结果　长波紫外线剂量依赖性地诱发表皮细胞的 DNA链断裂损伤 ,损伤的高峰出现在照射后 1h。而姜黄素可以拮抗长波紫外线引起的DNA链断裂 ,表现为彗星细胞百分比的降低和彗星尾巴长度的缩短 ,并有量效关系。结论　姜黄素可以预防长波紫外线照射引起的皮肤细胞 DNA链断裂损伤。     

西多福韦对HeLa细胞增殖抑制作用的研究

目的 研究抗病毒药西多福韦(CDV)对HeLa细胞的增殖抑制作用.方法 采用MTT法分析CDV和阳性对照药顺铂(DDP)对HeLa细胞增殖活力的影响;利用细胞集落形成实验和Giemsa染色技术,观察CDV和DDP导致HeLa细胞凋亡情况;利用流式细胞仪观察CDV和DDP对细胞凋亡及细胞周期影响.结果 MTT法和细胞集落实验结果显示CDV能明显抑制HeLa细胞的增殖活性;流式细胞仪测定显示CDV和DDP使HeLa细胞阻滞于细胞周期的S期,并诱导HeLa细胞发生凋亡.结论 CDV对HeLa细胞具有明显的增殖抑制作用,并能诱导细胞凋亡,CDV有可能成为宫颈癌治疗的另一策略.     

Atorvastatin effect on high-density lipoprotein-associated paraoxonase activity and oxidative DNA damage

Objective High-density lipoprotein (HDL)-associated antioxidant paraoxonase (PON) may reduce low-density lipoprotein (LDL) oxidation and prevent atherosclerosis. The aim of this present study was to investigate the effect of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor atorvastatin on hydrogen-peroxide-induced DNA damage by comet assay and the correlation between oxidative DNA damage and antioxidant PON activity. Methods Thirteen type-II/a hyperlipidemic patients were enrolled in the study. We examined the effect of 10 mg/day atorvastatin treatment on lipid levels and the degree of DNA damage in lymphocytes separated from hyperlipidemic patients, nitric oxide (NO), thiobarbituric acid-reactive substances (TBARS), PON levels and activity. Results After 6 months, atorvastatin treatment significantly decreased serum cholesterol and LDL-cholesterol levels. The triglyceride level did not change, and there was no significant change in the HDL cholesterol level. The visual score characteristic to the degree of DNA damage in comet assay was significantly decreased, as well as the TBARS level, while the level of NO was non-significantly increased. PON activity and the PON/HDL ratio were significantly increased after atorvastatin treatment. There was a negative correlation between DNA damage and PON activity, as well as between DNA damage and the PON/HDL ratio before and after atorvastatin treatment. Conclusion These findings show that atorvastatin treatment favorably affected the lipid profile, increasing the activity of HDL-associated PON and decreasing the cytotoxic effect of oxidative stress.     

钾通道阻断剂部分抑制三氧化二砷诱导的HeLa细胞死亡

目的研究钾通道阻滞剂对三氧化二砷诱导的HeLa细胞死亡的作用。方法采用MTT法评价HeLa细胞的存活情况，采用膜片钳技术记录HeLa细胞的电压依赖性钾电流。结果As₂O₃(5 μmol·L^-1)孵育24 h引起显著的HeLa细胞死亡，As₂O₃(5 μmol·L^-1)孵育24 h后存活的细胞表现明显的电压依赖性钾电流密度增加。+80 mV电压下，As₂O₃(5 μmol·L^-1)孵育组电流密度(61±18) pA/10 pF(n=8)明显高于对照组(38±10) pA/10 pF(n=8,P<005)。As₂O₃诱导的HeLa细胞死亡可被共同孵育钾通道阻滞剂四氨基吡啶(3 mmol·L^-1)或四乙基铵(5 mmol·L^-1)所部分抑制。3 mmol·L^-1四氨基吡啶或5 mmol·L^-1四乙基铵对HeLa细胞无明显细胞毒作用。结论As₂O₃长期处理增加HeLa细胞的电压依赖性钾电流。As₂O₃诱导的HeLa细胞死亡可被钾通道阻滞剂四氨基吡啶或四乙基铵部分抑制。     

海洋生物面膜对中波紫外线氧化损伤的HeLa细胞的保护作用

目的 :探讨中波紫外线 (UVB)照射下天然生物膜对HeLa上皮细胞氧化损伤的保护作用。方法 :建立UVB(辐照强度为 7.15× 10 - 5J/cm2 )对HeLa上皮细胞氧化损伤模型。MTT法测定细胞活性 ;流式细胞仪测定细胞的凋亡和死亡率 ;酶法测定抗氧化酶 (GSH Px、CAT、SOD)活性和MDA含量及总抗氧化能力。结果 :离体条件下生物膜能①显著增加Hela细胞的活性 ;②提高细胞上清液中GSH Px、CAT、SOD的活性 ,降低MDA含量 ,且呈量效关系。结论 :生物膜在体外有抗UVB氧化损伤的作用。其作用机制可能是通过提高抗氧化酶含量、清除自由基等 ,减轻细胞损伤而发挥其保护作用的     

Prevention by vitamin E of DNA fragmentation and apoptosis induced by fumonisin B1 in C6 glioma cells

Fumonisin B₁ (FB₁), produced by the fungus Fusarium moniliforme, belongs to a class of sphingosine analogue mycotoxins that occur widely in the food chain. Epidemiological studies have associated consumption of Fusarium moniliforme-contaminated food with human oesophageal cancer in China and South Africa. FB₁ also causes equine leucoencephalomalacia. Evidence for induction of apoptosis by FB₁ was first obtained when C6 glioma cells were incubated with fumonisin B₁ (3–27 μM) causing DNA fragmentation profiles showing DNA laddering in gel electrophoresis and apoptotic bodies revealed by chromatin staining with acridine orange and ethidium bromide. Further confirmation experiments and comet assays have been performed under similar conditions. The results of the comet test show that FB₁ at 9 and 18 μM induces respectively 50 ± 2% and 40 ± 1% of cells with a comet with an increased tail length of 93 ± 9 μm and 102 ± 17 μm respectively. Under these concentrations, FB₁ induced DNA fragmentation and laddering and many apoptotic bodies. Pre-incubation of the cells with vitamin E (25 μM) for 24 h before FB₁ (18 μM) significantly reduced DNA fragmentation and apoptotic bodies induced by FB₁. Received: 30 August 1999 / Accepted: 18 January 2000     

Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity

Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 μM. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 μM. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30–60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present.     

In vitro induction of cytotoxicity and DNA strand breaks in CHO cells exposed to cypermethrin, pendimethalin and dichlorvos

The indiscriminate use of pesticides and herbicides to increase crop productivity has aroused a great concern among the environmental and health scientists due to their adverse effects in both target as well as non-target species. Although substantial information is available regarding their environmental and ecological impact, not much is known in regard to its toxicity in the mammalian system. Therefore a study was conducted for the assessment of cytotoxic and genotoxic effects of cypermethrin (Type II pyrethroid) dichlorvos (organophosphate) and pendimethalin (dinitroaniline herbicide) in Chinese hamster ovary (CHO) cells. CHO cells were exposed to 1 μM, 10 μM, 100 μM, 1000 μM, and 10,000 μM, cypermethrin, pendimethalin and dichlorvos for 3 h and cytotoxicity was assessed by MTT assay. Their genotoxic potential was also evaluated by Comet assay. The results demonstrate that dichlorvos and pendimethalin exhibited higher extent of cytotoxicity as compared to cypermethrin. A significant (p < 0.05) concentration dependent increase in DNA damage was observed with dichlorvos (0.01 μM and above) and pendimethalin (0.1 μM and above) as evident by Comet assay parameters viz., Olive tail moment (arbitrary units), tail DNA (%) and tail length (μM). Cypermethrin induced a significant (p < 0.05) DNA damage only at higher concentrations (1000 and 5000 μM). Our data indicates that these chemicals produce cytotoxicity and DNA damage in mammalian cells and should be used with caution.     

Shikonin regulates HeLa cell death via caspase-3 activation and blockage of DNA synthesis

Shikonin, isolated from the plant Lithospermum erythrorhizon Sieb. Et Zucc, inhibited tumor cell growth and induced cell death in various tumor cells, with 50% growth inhibition of human cervical cancer cells, HeLa, at 18.9±1.1 μmol L^-1. Treated with 40 μmol L^-1 shikonin, HeLa cells underwent marked apoptotic morphological changes such as a round shape, membrane blebbing and apoptotic bodies derived from the fragmented nuclei. Another hallmark of apoptosis, DNA fragmentation, was observed by gel electrophoresis. Shikonin (10 μmol L^-1) significantly blocked the transition from G1 to S phase in the HeLa cell cycle. Pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK) or caspase-8 inhibitor (Z-IETD-FMK) effectively inhibited shikonin-induced cell death, while caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-9 inhibitor (Z-LEHD-FMK) failed to affect cell death. Caspase-3 activity significantly increased within 12 h after shikonin treatment. Reduced expression of inhibitor of caspase-activated deoxyribonuclease (ICAD) after exposure to shikonin for 12 h suggests the resultant activation of caspase-activated deoxyribonuclease (CAD), leading to apoptosis.     

Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells

The aim of this work was to investigate genomic DNA damage in human oral cavity cells after exposure to different tobacco product preparations (TPPs). The oral carcinoma cell line 101A, gingival epithelial cells HGEC, and gingival fibroblasts HGF were exposed to TPM (total particulate matter from 3R4F cigarettes), ST/CAS (2S3 smokeless tobacco extract in complete artificial saliva), and NIC (nicotine). Treatments were for 24 h using TPM at its EC-50 doses, ST/CAS and NIC at doses with equi-nicotine units, and high doses for ST/CAS and NIC. Comet assays showed that TPM, but not ST/CAS or NIC, caused substantial DNA breaks in cells; only the high ST/CAS dose caused weak DNA damage. These results were confirmed by immunofluorescence for γ-H2AX protein. These data revealed that the combusted TPP caused substantial DNA damage in all cell types, whereas the two non-combusted TPPs exerted no or only minimal DNA damage. They support epidemiologic evidence on the relative risk associated with consumption of non-combusted versus combusted tobacco products, and help to understand potential genotoxic effects of such products on oral cavity cells.     

Adenovirus-mediated expression of UHRF1 reduces the radio- sensitivity of cervical cancer HeLa cells to γ-irradiation

Aim： An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa cells using adenovirus-mediated UHRF1 gene transfer （Ad5-UHRF1）.
Methods： Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA （siRNA）.
Results： Increased expression of UHRF1 by Ad5-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRF1-mediated radioresistance.
Conclusion： These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.     

关木通两种提取液对V79细胞DNA的损伤作用

目的 探讨关木通对DNA的损伤作用。方法 应用单细胞凝胶电泳技术（彗星实验）研究中药关木通的两种提取液对中国仓鼠肺成纤维细胞（V79）的DNA损伤情况。结果 关木通的两种提取液在一定浓度下均能导致V79细胞产生彗星现象，并且存在的剂量-反应关系。结论 提示关木通过V79细胞DNA具有损伤作用。     

螺旋藻多糖对HeLa细胞生长的影响

目的；研究螺旋藻多糖（polysaccharide from Spirulina platensis,PSP)对体外培养的HeLa细胞生长的影响。方法：MTT法测定螺旋藻多糖的抗肿瘤活性；光镜观察螺旋藻多糖对HeLa细胞形态学的影响；流式细胞术检测螺旋藻多糖对肿瘤细胞周期的影响。结果：随螺旋藻多糖浓度的增加，HeLa细胞存活率逐渐降低。抑制率逐渐增加；光镜下的观察显示，螺旋藻多糖作用24-48h后，细胞出现明显的形态学改变；流式细胞术证实螺旋藻多糖对肿瘤细胞存在周期特异性，使细胞发生G1期阻滞。结论：螺旋藻多糖抑制HeLa细胞的增殖，可能与该细胞发生G1期阻滞有关。