The in vitro genotoxic and cytotoxic effects of remeron on human peripheral blood lymphocytes

Remeron (Mirtazapine) is an antidepressant drug which exerts its action by blocking presynaptic α-2-adrenergic receptors and postsynaptic serotonin types 2 and 3 receptors. In this in vitro analysis, human peripheral blood lymphocytes was treated by remeron (10, 25, 40 and 55 μg/mL) for 24 hours and 48 hours periods, then it was attempted to study of genotoxic and cytotoxic effects of the substance on human peripheral blood lymphocytes by some tests such as sister chromatid exchange (SCE), chromosomal abnormalities (CA) and micronucleus (MN) tests. Also proliferating effect of the substance was investigated. Remeron didn’t significantly cause chromosomal abnormalities and sister chromatid exchange while caused micronucleus at 40 μg/mL concentration and 24?h periodic time and increased proliferation index of the both 24 and 48 hours treated cells was decreased in a concentration manner. Also, exposing to the remeron for 24 and 48 hours leaded to a decrease in mitotic index and nucleus division index in the cells by concentration dependent manner. These findings showed that remeron did not have significantly genotoxic effects on human peripheral blood lymphocytes while it showed cytotoxic effects on the cells, which is the first report on genotoxic and cytotoxic properties of remeron.     

CIK细胞杀瘤实验前后细胞表型与细胞毒性相关性研究

目的为了分析体外细胞因子诱导培养CIK细胞在体外杀瘤实验中的细胞表型变化与其杀瘤活性的相关性及为临床过继免疫治疗提供实验依据。方法采用体外诱导方法扩增培养正常人外周血淋巴细胞及单个核细胞,用CCK-8试剂检测培养14d的CIK细胞对SPC-A-1和BGC-823肿瘤细胞的细胞毒活性,应用流式细胞术测定杀瘤实验前后CD3＋等6种不同表型细胞百分率的变化。结果在与效靶比成正比的条件下,体外实验CIK细胞对两种肿瘤细胞的杀伤率都与CD3＋CD4＋,CD3＋CD4-CD8-细胞比例变化呈正相关（P〈0.05）,都与CD3＋CD25＋,CD3＋CD28＋,CD3＋CD25＋CD28＋细胞比例变化呈负相关（P〈0.05）,对SPC-A-1细胞的杀瘤率与CD3＋CD16＋CD56＋呈正相关（P〈0.05）。结论本研究表明靶细胞抗原在活化细胞中起重要作用,测定培养细胞活化相关表型可以间接监测其杀瘤能力,为临床CIK细胞过继免疫治疗的应用提供实验依据。     

Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil

The induction of micronuclei and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analyzed in the erythrocytes of peripheral blood and cephalic kidney of turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua), treated with crude oil (Statfjord B, Norway) and with nonylphenol. Significant increase in MN was observed in turbot kidney and blood after exposure to 30 ppb of nonylphenol, 0.5 ppm of oil, and after co-exposure to 0.5 ppm of oil spiked with additional mixture of alkylphenols and PAHs (P varied between 0.0054 and <0.0001). The induction of micronuclei was observed only in cod kidney after exposure to spiked oil (P=0.0317). Significant inter-specific differences after the exposure to 0.5 ppm of oil (P=0.0385) and after treatment with spiked oil (P=0.0067) were observed. In turbot cephalic kidney, the elevated levels of bi-nucleated cells were observed in all treatment groups (P values varied in a range from 0.05 to 0.0025) while the increase in cells with nuclear buds was noted after the exposure to 0.5 ppm of oil (P=0.05). The fragmented-apoptotic cells appeared after the exposure to nonylphenol (P=0.0039) and to spiked oil (P<0.0001). In turbot blood, only the significant induction in nuclear buds was detected. Statistically significant inter-tissue differences were found only in the induction of fragmented-apoptotic cells after the exposure to nonylphenol and to spiked oil.     

镍,铅,汞化合物体外诱发人淋巴细胞（UDS）的检测

用人外周血淋巴细胞的非程序ＤＮＡ合成对镍、铅、汞化合物进行了测试，结果表明，这三种重金属都具有诱发ＵＤＳ的活性，并提示，受试重金属化合物达到一定浓度后，ＵＤＳ的ｃｐｍ值不再随剂量增高而增高，似乎在一定实验条件下ＵＤＳ的修复合成能力有一定限度。     

海带岩藻-半乳聚糖硫酸酯对体外培养的人外周血淋巴细胞的影响

目的:检测海带岩藻-半乳聚糖硫酸酯（Fucoidan-Galactosan Sulfate, FGS）对体外培养的人外周血淋巴细胞增殖及亚群分化的影响。方法:本实验采用体外培养人外周血淋巴细胞的方法,并利用流式细胞仪检测FGS的药物活性。结果:FGS各剂量组（6.25～100μg·mL-1）中CD3+、CD4+、CD8+及CD19+、CD25+高于对照组,且CD4+/CD8+值也比对照组有所提高。结论:FGS可促进人外周血T、B细胞的增殖分化,提高IL-2R的含量。     

Analysis of sister chromatid exchange and micronuclei in peripheral blood lymphocytes of nurses handling cytostatic drugs.

The genotoxic effect of occupational exposure of 20 nurses who handled cytostatic drugs in medical oncology and haematology units was evaluated by micronucleus and sister chromatid exchange test. The duration of employment in the units and of exposure to cytostatics ranged from 1 to 31 years. The exposed nurses manifested an increase in cells with micronuclei as compared to the control group (P < 0.05). Nurses exposed to cytostatic drugs for 20-31 years showed a higher frequency of micronuclei (P < 0.05), whereas there was no difference in frequencies between the control group and the group exposed for 1-14 years (P > 0.05). The influence of the exposure period proved to be a significant parameter for the micronucleus test. No statistically significant differences were observed in sister chromatid exchange (P > 0.05).     

The in vitro effect of trimethoprim on blastogenic transformation of human peripheral blood lymphocytes.

Trimethoprim (TP) at concentrations of 1 × 10^?6 to 1 × 10^?3 M inhibits the incorporation of thymidine, uridine, formate, and leucine by human lymphocytes undergoing blastogenic transformation induced by conconavalin A. TP does not alter cell viability, as measured by trypan blue exclusion, or cell numbers. An effect of TP on dihydrofolate reductase, such as is seen with methotrexate, is insufficient to explain the inhibitory effects of TP since an in vivo estimation of dihydrofolate reductase activity, utilizing deoxyuridine incorporation into deoxyribonucleic acid, is not inhibited. Also the observed inhibition of precursor incorporation is not fully reversed by either folate or folinate. TP did not alter the percentage of cells transforming but reduced the number of autoradiographically-labelled lymphoblasts. Immunosuppression by TP in vivo may be associated with these findings.     

In vitro investigation of the genotoxic and cytotoxic effects of thiacloprid in cultured human peripheral blood lymphocytes

Thiacloprid, a neonicotinoid insecticide, is widely used for controlling various species of pests on many crops. The potential genotoxic effects of thiacloprid on human peripheral blood lymphocytes (PBLs) were investigated in vitro by the chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and cytokinesis‐block micronucleus (MN) assays. The human PBLs were treated with 75, 150, and 300 μg/mL thiacloprid in the absence and presence of an exogenous metabolic activator (S9 mix). Thiacloprid increased the CAs and SCEs significantly at all concentrations (75, 150, and 300 μg/mL) both in the absence and presence of the S9 mix and induced a significant increase in MN and nucleoplasmic bridge formations at all concentrations for 24 h and at 75 and 150 μg/mL for 48‐h treatment periods in the absence of the S9 mix; and at all concentrations in the presence of the S9 mix when compared with the control and solvent control. Thiacloprid was also found to significantly induce nuclear bud (NBUD) formation at 300 μg/mL for 24 h and at 150 μg/mL for 48‐h treatment times in the absence of the S9 mix and at the two highest concentrations (150 and 300 μg/mL) in the presence of the S9 mix. Thiacloprid significantly decreased the mitotic index, proliferation index, and nuclear division index for all concentrations both in the absence and presence of the S9 mix. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 631–641, 2014.     

Reduction of chrysotile asbestos-induced genotoxicity in human peripheral blood lymphocytes by garlic extract

Asbestos fibers are well known environmental carcinogen, however, the underlying mechanisms of their action have still not clearly been identified. Asbestos is capable of depleting glutathione and generating reactive oxygen species (ROS), which are important mediators of damage in biological system. Asbestos-induced mutagenecity, may be mediated by the generation. It is known that a number of scavengers and antioxidants attenuate asbestos-induced ROS release. Furthermore, it is known that garlic, contains numerous sulfur compounds and glutathione precursors which act as antioxidants and also demonstrate anticarcinogenic properties. The aim of this study was to investigate whether garlic extract has any influence on asbestos-mediated genotoxicity. As an assay system, we applied the micronucleus assay, sister chromatid exchanges, and chromosomal aberrations with human peripheral blood lymphocytes, which has already been used to analyze the genotoxicity of asbestos fibers. Our results indicate that garlic extract, when administered to the lymphocytes cell culture simultaneously with chrysotile reduced the rates of micronucleus formation, sister chromatid exchanges, and chromosomal aberrations significantly. We conclude that garlic extract may be an efficient, physiologically tolerable quencher of asbestos-mediated genotoxicity.     

慢性乙型肝炎患者外周血淋巴细胞穿孔素的表达

目的：了解慢性乙型肝炎患者外周血淋巴细胞（PBL）穿孔素（PFP）的表达情况及其与疾病的关系。方法：用抗人PFP单克隆抗体，采用免疫组化染色法，观察慢性乙型肝炎患者PBL中总的PFP阳性细胞的百分率，结果：慢性乙型肝炎PBL中PFP表达与正常对照组之间存在明显差异，结论：乙型肝炎病毒感染慢性化与机体PBL中PFP的表达水平低下密切相关。     

Genotoxic and cytotoxic effects of doxycycline in cultured human peripheral blood lymphocytes

Doxycycline (DOX) is a broad-spectrum tetracycline antibiotic used in the treatment of many infections. In this study, the genotoxic and cytotoxic effects of DOX in cultured human peripheral blood lymphocytes were investigated by measuring chromosome aberrations (CAs), cytokinesis-block micronucleus (CBMN) assay, mitotic index (MI), and nuclear division index (NDI). Cultures were treated with DOX at three concentrations (2, 4, and 6 μg/mL) for 48 hours. Mitomycin C (MMC) was used as a positive control. All the tested concentrations of DOX for MI and the higher concentrations (4 and 6 μg/mL) for NDI significantly decreased mitotic activity. However, there are no significant differences between negative control and all the tested concentrations of DOX for CA and MN frequencies. In conclusion, our results indicate that DOX has a cytotoxic effect, but not a genotoxic effect, on human peripheral blood lymphocyte cultures. Further detailed studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to different types of cancer cells is investigated in detail, it may also have been used as an antitumoral drug.     

Genotoxic and cytotoxic effects of doxycycline in cultured human peripheral blood lymphocytes

Doxycycline (DOX) is a broad-spectrum tetracycline antibiotic used in the treatment of many infections. In this study, the genotoxic and cytotoxic effects of DOX in cultured human peripheral blood lymphocytes were investigated by measuring chromosome aberrations (CAs), cytokinesis-block micronucleus (CBMN) assay, mitotic index (MI), and nuclear division index (NDI). Cultures were treated with DOX at three concentrations (2, 4, and 6 µg/mL) for 48 hours. Mitomycin C (MMC) was used as a positive control. All the tested concentrations of DOX for MI and the higher concentrations (4 and 6 µg/mL) for NDI significantly decreased mitotic activity. However, there are no significant differences between negative control and all the tested concentrations of DOX for CA and MN frequencies. In conclusion, our results indicate that DOX has a cytotoxic effect, but not a genotoxic effect, on human peripheral blood lymphocyte cultures. Further detailed studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to different types of cancer cells is investigated in detail, it may also have been used as an antitumoral drug.     

Studying the impact of S9 on cyto-genotoxicity of cigarette smoke in human peripheral blood lymphocytes in vitro

In present study, human lymphocytes were exposed to cigarette smoke condensates (CSCs) at the doses of 25, 50, 75, 100 and 125 μg ml⁻¹ with and without S9, and the cyto-genotoxic effects were detected with CCK-8, cell apoptosis and micronucleus assays. DNA repair kinetics was observed with comet assay. Our results indicated that the cell viability decreased with CSCs doses, the percentages of apoptosis cell and the frequencies of micronuclei increased with CSCs doses, and DNA damage of human lymphocytes induced by CSCs could be basically repaired within 240 min. However, the cytotoxicity induced by CSCs +S9 was significantly lower than that induced by CSCs −S9 in CCK-8 and cell apoptosis assays, and the DNA repair speed in +S9 group was quicker than that in −S9 group. In conclusion, S9 may affect not only the cyto-genotoxicity of CSCs but also the repair process of DNA damage induced by CSCs in lymphocytes.     

动物及健康人肠道共生的大肠埃希菌耐药性及产生原因分析

目的了解目前动物和人肠道共生的大肠埃希菌耐药性,分析其产生的原因。方法从甘肃、湖北、北京、山东、四川等地相对封闭的养殖场及养殖场附近的健康人群采集鸡、猪、鱼、人粪便样本,分离大肠埃希菌。用AP I20E鉴定条鉴定怀疑为大肠埃希菌的菌种,采用KB纸片法检测生化鉴定为大肠埃希菌的菌株的耐药性,利用WHONET 5.3软件进行药敏试验数据分析。用脉冲场凝胶电泳技术检测具有相似耐药谱型的大肠埃希菌菌株间的同源性。结果①共收集571株大肠埃希菌,其中从海水养殖鱼类和淡水养殖鱼类分离31株,另从海水养殖鱼类和淡水养殖鱼类分离嗜水气单胞菌株57株;②来源鸡的大肠埃希菌对所有检测的抗菌药物的耐药率最高;③除β-内酰胺类抗生素和阿米卡星外,鸡、猪和人来源菌株的耐药率表现为高、中、低现象,对老的一些抗菌药物和喹诺酮类抗菌药物尤其明显;不同地区分离株的耐药性也有较大的差异;④本次调查首次在国内养鸡场发现产ESBL大肠埃希菌,并且非常多见;⑤嗜水气单胞菌和大肠埃希菌的耐药性有较大的差异,47株耐药谱型相近的菌株中发现了三组基因水平同源性菌株(相似度大于95%)。结论①从不同地区、不同种类动物分离菌株的耐药性不同,与抗生素的使用情况相关。目前我国家禽养殖业滥用喹诺酮类和β-内酰胺类尤其是三代头孢菌素类抗生素的现象较为普遍,应该加以严格控制;②不同菌种的生物学特性不同,导致耐药性不同;③同源性分析发现耐药菌株可在同一种类动物间传播,不同类动物的大肠埃希菌之间可能存在耐药基因的水平传播;④应当加强养殖动物分离的大肠埃希菌耐药性监测。     

Chromosomal aberrations, micronuclei and nuclear buds induced in human lymphocytes by 2,4-dichlorophenoxyacetic acid pesticide formulation

Pesticides of worldwide application are used in agriculture in vast amounts each year, of which herbicides are the most prominent class. Phenoxyacetic herbicides constitute one of the largest groups of herbicides sold in the world. Among them, for many years 2,4-dichlorophenoxyacetic acid (2,4-D) has been the one most used. In this study we used Deherban A, a commercial formulation of 2,4-D to determine its possible genotoxic effect on human lymphocytes in vitro by chromosomal aberration analysis and micronucleus assay including the scoring of nuclear buds. Two different concentrations of pesticide formulation were used so that final concentrations of 2,4-D were 0.4 and 4 microg/ml, both in the presence and in the absence of the liver microsomal fraction as metabolic activator. Both concentrations of pesticide caused an increase in chromatid and chromosome breaks, number of micronuclei and number of nuclear buds. Presence of the S9 mix additionally elevated the number of chromatid breaks and micronuclei in treated lymphocytes.     

体外小鼠、豚鼠、犬及人血液对梭曼的解毒作用

目的　研究不同种属动物及人血液对梭曼的解毒能力与血液中几种梭曼解毒酶活性的关系。方法　测定血液中梭曼残留浓度及解毒酶活性。结果　小鼠、豚鼠、犬、人血浆对梭曼的解毒能力要明显高于其自身红细胞的解毒能力 ,血浆对梭曼的解毒能力依小鼠、豚鼠、犬、人的顺序由高到低依次排列。啮齿类动物血液中羧酸酯酶 (CaE)活性位点数目多且与梭曼结合快 ,因此在梭曼解毒中可发挥重要作用。犬、人血液中乙酰胆碱酯酶 (AChE)在梭曼解毒中占据了重要的解毒地位 ,犬、人血液中CaE基本没有参与梭曼解毒。结论　血液解毒能力的种属差异与种属间解毒酶结合位点数量的多少及梭曼与酶的结合速率密切相关     

In vitro effect of the antimalarial drug proguanil hydrochloride on viability and DNA damage in human peripheral blood lymphocytes

This study aimed to evaluate the effect of proguanil, a chemical substance used for treatment and prevention of malaria on viability and DNA integrity in human lymphocytes in vitro. Two different concentrations of proguanil obtained from the plasma concentrations were used: 130 ng/ml used for prophylactic treatment and 520 ng/ml used in treatment of malaria. Testing was done with and without metabolic activation. Viability of lymphocytes decreased in time and dose dependent manner. Comet assay parameters showed similar effects, indicating that some damage to DNA molecule can occur. Frequency of sister chromatid exchanges did not show significant deviation from the control samples. As for the proliferation kinetics no significant changes were noticed. Since majority of DNA damaging effect is induced after metabolic activation it is to be concluded that activity of proguanil is dependent upon the active metabolite cycloguanil and that monitoring should be conducted especially among frequent travellers.     

In vitro effect of the antimalarial drug proguanil hydrochloride on viability and DNA damage in human peripheral blood lymphocytes

This study aimed to evaluate the effect of proguanil, a chemical substance used for treatment and prevention of malaria on viability and DNA integrity in human lymphocytes in vitro. Two different concentrations of proguanil obtained from the plasma concentrations were used: 130ng/ml used for prophylactic treatment and 520ng/ml used in treatment of malaria. Testing was done with and without metabolic activation. Viability of lymphocytes decreased in time and dose dependent manner. Comet assay parameters showed similar effects, indicating that some damage to DNA molecule can occur. Frequency of sister chromatid exchanges did not show significant deviation from the control samples. As for the proliferation kinetics no significant changes were noticed. Since majority of DNA damaging effect is induced after metabolic activation it is to be concluded that activity of proguanil is dependent upon the active metabolite cycloguanil and that monitoring should be conducted especially among frequent travellers.     

Cytotoxic and immunomodulative effects of flunitrazepam, zipeprol and trihexyphenidyl on human peripheral blood lymphocytes

The cytotoxic effects of flunitrazepam, zipeprol and trihexyphenidyl on cell viability of peripheral blood mononuclear cells from healthy volunteers were studied at concentrations from 10(-2) to 10(-8) M by 2 colorimetric in vitro assays: the neutral red uptake assay and thiazolyl blue tetrazolium bromide assay. All tested drugs of abuse were non-cytotoxic at concentrations lower than 10(-5) M. Possible immunomodulative effects of these substances were evaluated through phytohemagglutinin induced lymphocyte proliferation ([3H]-thymidine DNA incorporation assay) and by a 51Cr release natural killer assay. The 3 drugs studied produced statistically significant immunomodulative alterations on both immunological parameters.