In vitro genoprotective and genotoxic effect of nicotine on human leukocytes evaluated by the comet assay

Abstract

The comet assay was used to measure the DNA damage induced in vitro by nicotine in human leukocytes as the extent of DNA migration in the comet head area, tail length, percent DNA in the tail, and Olive tail moment. Samples of whole blood were collected and blood cells were challenged with acute doses of 0.1, 1 and 10?µM of (?)-nicotine for 60 minutes. We found that nicotine treatment had dose-dependent effects on the level of DNA damage. At 1 and 10?µM of nicotine, both Olive tail moment and percent DNA in the tail significantly increased (p?<?0.001), compared to the control. In the presence of 10?µM of nicotine, the shortest tail length and the smallest head area were detected. At a concentration of 0.1?µM, surprisingly, DNA damage detected by the comet assay was lower than in the control, which was proved by the observed significantly (p?<?0.001) lower Olive tail moment and percent DNA in the tail as well as larger head area. The results suggest that nicotine, at a reasonably low concentration (0.1?µM), comparable to those found in the blood of habitual smokers, may have a protective effect, whereas higher doses of nicotine (1 and 10?µM) are genotoxic. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine is discussed.     

Interaction of platinum agents,cisplatin, carboplatin and oxaliplatin against albumin in vivo rats and in vitro study using inductively coupled plasma‐mass spectrometory

The protein binding rates (PBR) of platinum‐containing agents cisplatin (CDDP), carboplatin (CBDCA) and oxaliplatin (L‐OHP) have been reported as 98%, 25–50% and 98%, respectively. To investigate the protein‐binding properties of albumin with cisplatin, carboplatin and oxaliplatin, inductively coupled plasma mass spectrometry (ICP‐MS) was used to measure their plasma concentration in rats over time. The study also examined the effects of cisplatin, carboplatin and oxaliplatin‐binding on albumin in vitro, using CD spectrometry and native‐polyacrylamide gel electrophoresis (native PAGE). The ratios of PBR to irreversible PBR, of cisplatin and oxaliplatin were 98%:98% and 90%:87%, respectively, indicating a higher affinity for irreversible binding with albumin. That of carboplatin was 25%:10%, indicating 60–70% reversible binding with albumin. The plasma protein binding rate concentrations of cisplatin, carboplatin and oxaliplatin after in vivo administration were 96%, 15% and 80%, respectively. The CD spectrometry of albumin was unaffected by cisplatin, carboplatin and oxaliplatin binding. Though similar protein binding rates were observed with oxaliplatin and cisplatin, oxaliplatin had a higher mobility rate during PAGE. It was confirmed that the binding of cisplatin and oxaliplatin with albumin affected its electric charge but not the structure. In conclusion, cisplatin and oxaliplatin bind irreversibly with albumin in plasma and may irreversibly interact with tissue protein and/or DNA. The difficulties involved with predicting the tissue concentrations of cisplatin and oxaliplatin from their plasma concentration inhibits their therapeutic drug monitoring. On the contrary, carboplatin, like some generic drugs, reversibly binds to plasma proteins. It is, therefore, possible to conduct therapeutic drug monitoring for carboplatin.     

重组人肝细胞生长因子β链体外生物学活性的测定

目的　建立一种稳定灵敏的方法用于检测重组人肝细胞生长因子β链 (rhHGF β)对原代培养成年大鼠肝细胞的增生作用。方法　利用噻唑蓝 (MTT)比色方法在多种实验条件下对rhHGF β进行生物学活性检测。结果　对于体外原代培养成年大鼠肝细胞 ,rhHGF β可刺激肝细胞的增生 ,且有剂量依赖效应。 结论　确定了原代培养肝细胞检测rhHGF β生物活性的最佳条件方案     

In vitro aneugenic effects of the fungicide thiabendazole evaluated in human lymphocytes by the micronucleus assay

Thiabendazole is a benzimidazole-derived compound widely employed in agriculture as anthelmintic and fungicide. It is also used as a post-harvest fungicide for imported citrus fruits during transport and storage, and thus, it was found at high concentration in fruits and vegetables. Several studies have analyzed the potential genotoxic effect of thiabendazole on different prokaryotic and eukaryotic systems, but in many cases, results were contradictory. In the present study, the genotoxic potential of thiabendazole have been evaluated, by micronucleus assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 0.5, 5 and 50 μg/ml concentrations of the tested substance for 48 h at 37°C. Mitomycin C at final concentration of 0.01 μg/ml culture was used as a positive control. The results indicated that the thiabendazole significantly (P < 0.05) increased the micronucleus frequency compared with the negative control in all treatment concentrations, indicating a potential aneugenic hazard of thiabendazole in cultured human peripheral lymphocytes. The cytokinesis-block proliferation index value, however, was not decreased significantly compared with the negative control. Significant (P < 0.05) differences in the micronuclei frequency were also found between the lower dose (0.5 μg/ml) and the other two analyzed doses of thiabendazole. In contrast, no differences were found between 5 and 50 μg/ml of thiabendazole and between DMSO and negative control. Finally, control cultures treated with the known mutagen MMC showed a very consistent increase in MN with respect to the negative controls.     

铂类联合多西他赛一线治疗晚期食管癌

目的:本临床研究采用铂类(顺铂或卡铂)联合多西他赛(docetaxel)一线治疗晚期食管癌,探讨晚期食管癌的一线治疗及预后因素.方法:2006年8月至20HD8年10月,共有36例患者入选,其中27例采用顺铂(DDP)联合docetaxel(DT)方案进行治疗,9例采用卡铂(CBP)联合docetaxel(CT)方案进行治疗;DT方案:DDP 25 mg/m~2,第1～3天或第1～4天使用;docetaxel 75 mg/m~2,第1天使用,以上用法3周重复.CT方案:DT方案中把DDP替换为CBP AUC=5.结果:入选36例患者均能进行疗效和毒副反应评价,完全缓解(CR)1例;部分缓解(PR)17例,稳定(NC)10例,进展(PD)7例,总有效率为50.00%;DT、CT方案有效率分别为59.26%及22.22%(P=0.121).较常见的毒副作用为恶心呕吐,经对症支持治疗后均见好转.中位随访时间为8.70个月,全组36例患者中位生存期为10.50个月,95%的可信区间为7.36～13.64.COX单因素回归分析提示:性别、年龄、体重下降及治疗前血红蛋白对生存的影响均没有统计学意义(P＞0.05),而治疗前行为状态对生存的影响差异有统计学意义(P=0.036).结论:铂类联合多西他赛一线治疗晚期食管癌疗效好,毒副反应可耐受;治疗前行为状态是影响生存的独立因素,行为状态良好治疗后预后也较好.     

Genotoxicity of nicotine in cell culture of Caenorhabditis elegans evaluated by the comet assay

To assess the genotoxicity of nicotine, its DNA-damaging effect on Caenorhabditis elegans cells was tested with the alkaline single-cell microgel electrophoresis (comet) assay. The degree of DNA migration (a measure of possible DNA single-strand breaks, alkali-labile sites, and incomplete excision repair sites) was expressed as the head DNA%, tail length, and Olive tail moment. Large differences were found between experimental variants: 0, 1, 10, and 100?μM (-)-nicotine. At concentrations of 1 and 10?μM, no damages were detected by the comet assay, and the Olive tail moment and tail length were significantly lower than in the control (P?<?0.001). The highest head DNA% and the lowest tail length and Olive tail moment were observed in the presence of 1?μM of nicotine. At 100?μM of nicotine, a significant increase (P?<?0.001) was observed in Olive tail moment and tail length (up to 2.7- and 3-fold, respectively, compared to the control). The results are consistent with the lowest head DNA% among the three tested variants. This study demonstrated that nicotine treatment had dose-dependent effects on the level of DNA damage. Generally, a high dose of nicotine (100?μM) is genotoxic, while a reasonably low concentration has a protective effect. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine in C. elegans is discussed.     

肿瘤体外顺铂敏感性的影响因素

目的 研究肿瘤体外药敏试验中顺铂敏感性的影响因素.方法 回顾性分析138例卵巢癌和67例宫颈癌患者的MTT法肿瘤体外药敏试验顺铂敏感性数据,采用非条件logistic回归分析年龄、肿瘤病理类型和顺铂化疗史的间隔时间对体外顺铂敏感性的影响.结果 年龄、肿瘤病理类型和顺铂化疗史的间隔时间对体外顺铂敏感性的影响没有统计学意义(P=0.6556).结论 卵巢癌和宫颈癌患者体外顺铂敏感性具有较大的个体差异,由于回归系数的误差、样本数小及变量赋值等影响,使本研究得到阴性结果,但有顺铂化疗史的卵巢癌和宫颈癌患者仍然有体外顺铂敏感的可能性.     

血清中环丙沙星的微量微生物测定法

微量微生物法测定血清中环丙沙星浓度的各项方法学指标均符合要求，不需特殊的仪器和试剂，操作简便、取样量少。试验菌选用枯草或大肠杆菌，两菌的测定结果经统计学处理无显著差异（Ｐ＞０．０５）。适用于体内药物浓度监测。     

微生物法测定血浆中万古霉素血药浓度的研究

目的:探讨血浆中万古霉素浓度的微生物测定方法.方法:采用微生物测定法,测定万古霉素血药浓度,枯草芽胞杆菌作为试验菌.结果:万古霉素在1.0～60.0 μg·ml-1范围内,线性关系良好(r=0.9992);日内、日间相对标准误差在3.3%～4.3%.结论:用微生物法测定万古霉素血药浓度,简便、准确,可用于临床治疗药物浓度监测.     

微量微生物法测定血清中司帕沙星浓度

目的 建立血清中司帕沙星（ＳＰＬＸ）浓度微生物测定。方法 采用微量微生物法。选用枯草杆菌、大肠杆菌为实验菌种测定ＳＰＬＸ血药浓度。结果 测量枯草杆菌的回收率：（９８．７５～１０５．５０）％,ＲＳＤ为（１．３～５．４）％;大肠杆菌的回收率为（（９５．８８～１０４．００％）,ＲＳＤ为（１．０～６．１）％。结论 用微量生物法测定ＳＰＬＸ血药浓度,操作简便,成本低廉,样品用量少,精密度高,适用于体内药物浓     

Discrepancy between cytotoxicity and DNA interstrand crosslinking of carboplatin and cisplatin in vivo

We used the method of alkaline elution to compare quantitatively the DNA lesions produced by cisplatin and carboplatin in the AKR leukemia in vivo. These data were compared with cytotoxicity of each drug in the same animal model and in a solid tumor murine model (colon 26).DNA-protein and DNA-DNA interstrand crosslinks were formed in similar proportions by both drugs when peak values of crosslinking were compared. No clear difference in the rate of formation of both types of crosslinks could be observed between these drugs.On a molar basis a 3- to 4-fold more carboplatin had to be given to obtain equivalent frequencies of both types of crosslinks. In contrast, to obtain equitoxicity in the same animal tumor model, 13 fold higher doses of carboplatin had to be given. This difference in cytotoxicity between both drugs is comparable to the difference measured in colon 26 in vivo (16 fold). Both values are in the range of literature data (10–25 fold) dealing with the relative potency of cisplatin and carboplatin in murine tumor models.Abbreviations DIC DNA-DNA Interstrand Crosslinks - DPC Protein-DNA Crosslinks - EDTA Ethylene-Diammine-Tetra-Acetic Acid - PK Proteinase K - PSA Puck's Saline A - SSB Single Strand Breaks - ILS Increase in Life Span - Carboplatin-Cis-(diammino)(1,1-cyclobutane-dicarboxylato)-Platinum (II), Cisplatin Cis-Diamminedichloroplatinum (II) Supported by an award from the Belgian Work against Cancer.American Cancer Society Cancer Development Award Recipient.     

吉西他滨联合卡铂诱导NK/T细胞淋巴瘤细胞株凋亡的研究

目的:研究吉西他滨联合卡铂对NK/T细胞淋巴瘤细胞株(SNK 6)细胞增殖及凋亡的影响。方法:采用细胞增殖-毒性检测法(CCK-8)检测吉西他滨、卡铂、顺铂对SNK 6细胞增殖的影响,算出各自半数抑制浓度(IC 50)值;再用吉西他滨与卡铂或顺铂做联合实验,用金氏公式计算Q值,评价联合用药效应。应用流式细胞仪检测不同药物组对细胞凋亡的影响。蛋白免疫印迹法(Western Blotting)检测各药物组Cleaved-caspase-3、B淋巴细胞瘤-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)等凋亡蛋白的表达水平。结果:吉西他滨、卡铂、顺铂对SNK 6细胞均有较好的抑制作用;联合用药中,吉西他滨+卡铂组与吉西他滨+顺铂组对SNK 6细胞抑制作用相当,两药联合为相加作用。吉西他滨+卡铂组凋亡率稍低于吉西他滨+顺铂组,但差异无统计学意义(P>0.05)。吉西他滨可上调Cleaved-caspase-3蛋白、Bax蛋白的表达水平,下调Bcl-2蛋白的表达水平(P<0.05),吉西他滨与卡铂或顺铂联合时效果更加显著。结论:在体外环境下,吉西他滨联合卡铂可抑制SNK 6细胞增殖并诱导其凋亡,且一定浓度的吉西他滨联合卡铂对SNK 6细胞株的凋亡率与吉西他滨联合顺铂相似。     

Nicotine derived genotoxic effects in human primary parotid gland cells as assessed in vitro by comet assay,cytokinesis-block micronucleus test and chromosome aberrations test

Genotoxic effects of nicotine were described in different human cells including salivary gland cells. Based on the high nicotine concentration in saliva of smokers or patients using therapeutic nicotine patches, the current study was performed to evaluate the genotoxic potential of nicotine in human salivary gland cells.Therefore, primary salivary gland cells from 10 patients undergoing parotid gland surgery were exposed to nicotine concentrations between 1 μM and 1000 μM for 1 h in the absence of exogenous metabolic activation. The acinar phenotype was proven by immunofluorescent staining of alpha-amylase. Genotoxic effects were evaluated using the Comet assay, the micronucleus test and the chromosome aberration test. Cytotoxicity and apoptosis were determined by trypan blue exclusion test and Caspase-3 assay.Nicotine was able to induce genotoxic effects in all three assays. The chromosome aberration test was the most sensitive and increases in numerical and structural (chromatid-type and chromosome-type) aberrations were seen at ⩾1 μM, whereas increases in micronuclei frequency were detected at 10 μM and DNA damage as measured in the Comet assay was noted at >100 μM. No cytotoxic damage or influence of apoptosis could be demonstrated.Nicotine as a possible risk factor for tumor initiation in salivary glands is still discussed controversially. Our results demonstrated the potential of nicotine to induce genotoxic effects in salivary gland cells. These results were observed at saliva nicotine levels similar to those found after oral or transdermal exposure to nicotine and suggest the necessity of careful monitoring of the use of nicotine in humans.     

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.     

铂类抗肿瘤药物的研发进展及市场情况

近年来,铂类抗肿瘤药物的临床研究进展较快,其抗肿瘤谱广,抗肿瘤活性增强,不良反应降低,已成为目前有关抗肿瘤药物研发的重要领域。新的铂类抗肿瘤药物将从那些在临床研究中显示出低毒性、抗肿瘤谱广、与现有药物无交叉耐药性的化合物中产生。本文就其作用机制、国内外上市开发现状、国内外销售情况及国内研究开发进展等进行综述。     

An in vitro human cell-based assay to rank the relative immunogenicity of proteins.

A method to rank proteins based on their relative immunogenicity has been devised. A statistical analysis of peptide-specific responses in large human donor pools provides a structure index value metric that ranked four industrial enzymes in the order determined by both mouse and guinea pig exposure models. The ranking method also compared favorably with human sensitization rates measured in occupationally exposed workers. Structure index values for other proteins known to cause immune responses in humans were also determined and found to be higher than the value determined for human beta2-microglobulin. Using values from known immunogenic and putative nonimmunogenic proteins, a cut-off value was established. The structure index value calculation provides a comparative method to predict subsequent immunogenicity on a human population basis without the need to use animal models. Information provided by this assay can be used in the early development of protein therapies and other protein-based applications to select or create reduced immunogenicity variants.     

国产卡铂和顺铂在兔体内的药物动力学

本文以石墨炉原子吸收分光光度法为测定方法,对国产卡铂和顺铂在家兔体内的药物动力学进行了初步的研究和比较。本法中卡铂和顺铂在0～20μg/ml范围内药物血浆浓度同吸收峰高呈线性关系,相关系数r>0.99(P相似文献   

In vitro antitumor effect of recombinant human tumor necrotizing factor on cultured human cancer cell lines and freshly isolated lung cancer cells by the human tumor clonogenic assay

Summary In vitro antitumor effects of human recombinant tumor necrotizing factor (rH-TNF) were examined against nine lung cancer cell lines including six non small and three small cell lung cancer, four stomach cancer cell lines and 30 freshly isolated lung cancer cell samples by the human tumor clonogenic assay. rH-TNF did not show any inhibitory effect on the colony formations of lung and stomach cancer cell lines, except for PC10 established from squamous cell carcinoma even at the high concentration. The overall response rate of fresh material was 11.5%. The colony formations of only two materials from 20 patients without prior chemotherapy were significantly suppressed by rH-TNF in vitro. Three specimens of adenocarcinoma exhibited more than 70% decrease in colony number by treating with 100 and 1000 u/ml of rH-TNF resulting in the response rate of 15.8% (3/19). From these results, it can be concluded that rH-TNF has modest direct cytotoxic effect on lung cancer, and additional study against adenocarcinoma of the lung might be warranted.     

Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes

Excessive release of stress hormone adrenaline is accompanied by generation of reactive oxygen species which may cause disruption of DNA integrity leading to cancer and age-related disorders. Phenolic-rich plant product dry olive leaf extract (DOLE) is known to modulate effects of various oxidants in human cells. The aim was to evaluate the effect of commercial DOLE against adrenaline induced DNA damage in human leukocytes by using comet assay. Peripheral blood leukocytes from 6 healthy subjects were treated in vitro with three final concentrations of DOLE (0.125, 0.5, and 1 mg/mL) for 30 min at 37 °C under two different protocols, pretreatment and post-treatment. Protective effect of DOLE was assessed from its ability to attenuate formation of DNA lesions induced by adrenaline. Compared to cells exposed only to adrenaline, DOLE displayed significant reduction (P < 0.001) of DNA damage at all three concentrations and under both experimental protocols. Pearson correlation analysis revealed a significant positive association between DOLE concentration and leukocytes DNA damage (P < 0.05). Antigenotoxic effect of the extract was more pronounced at smaller concentrations. Post-treatment with 0.125 mg/mL DOLE was the most effective against adrenaline genotoxicity. Results indicate genoprotective and antioxidant properties in dry olive leaf extract, strongly supporting further explorations of its underlying mechanisms of action.