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1.
Fenech M Bonassi S Turner J Lando C Ceppi M Chang WP Holland N Kirsch-Volders M Zeiger E Bigatti MP Bolognesi C Cao J De Luca G Di Giorgio M Ferguson LR Fucic A Lima OG Hadjidekova VV Hrelia P Jaworska A Joksic G Krishnaja AP Lee TK Martelli A McKay MJ Migliore L Mirkova E Müller WU Odagiri Y Orsiere T Scarfì MR Silva MJ Sofuni T Surralles J Trenta G Vorobtsova I Vral A Zijno A Suralles J;HUman MicroNucleus project 《Mutation research》2003,534(1-2):45-64
One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations. 相似文献
2.
Holland N Bolognesi C Kirsch-Volders M Bonassi S Zeiger E Knasmueller S Fenech M 《Mutation research》2008,659(1-2):93-108
The micronucleus (MN) assay in exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. This overview has concluded that although MN assay in buccal cells has been used since the 1980s to demonstrate cytogenetic effects of environmental and occupational exposures, lifestyle factors, dietary deficiencies, and different diseases, important knowledge gaps remain about the characteristics of micronuclei and other nuclear abnormalities, the basic biology explaining the appearance of various cell types in buccal mucosa samples and effects of diverse staining procedures and scoring criteria in laboratories around the world. To address these uncertainties, the human micronucleus project (HUMN; see http://www.humn.org) has initiated a new international validation project for the buccal cell MN assay similar to that previously performed using human lymphocytes. Future research should explore sources of variability in the assay (e.g. between laboratories and scorers, as well as inter- and intra-individual differences in subjects), and resolve key technical issues, such as the method of buccal cell staining, optimal criteria for classification of normal and degenerated cells and for scoring micronuclei and other abnormalities. The harmonization and standardization of the buccal MN assay will allow more reliable comparison of the data among human populations and laboratories, evaluation of the assay's performance, and consolidation of its world-wide use for biomonitoring of DNA damage. 相似文献
3.
M. J. Ramírez J. Surralls S. Puerto A. Creus R. Marcos 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》1999,440(2):567
The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of γ-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed. 相似文献
4.
Phenotype frequencies of autosomal minor histocompatibility antigens display significant differences among populations 总被引:1,自引:0,他引:1
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Spierings E Hendriks M Absi L Canossi A Chhaya S Crowley J Dolstra H Eliaou JF Ellis T Enczmann J Fasano ME Gervais T Gorodezky C Kircher B Laurin D Leffell MS Loiseau P Malkki M Markiewicz M Martinetti M Maruya E Mehra N Oguz F Oudshoorn M Pereira N Rani R Sergeant R Thomson J Tran TH Turpeinen H Yang KL Zunec R Carrington M de Knijff P Goulmy E 《PLoS genetics》2007,3(6):e103
Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen–matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen–matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated. 相似文献
5.
The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of gamma-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed. 相似文献
6.
A decrease in the cytokinesis-block proliferation index (CBPI) or replication index (RI) is routinely used to determine cytotoxicity of a test compound and therefore the choice of its appropriate test concentration for the in vitro micronucleus (MN) test conducted in the presence of cytochalasin B. As a number of laboratories prefer to conduct the in vitro MN test in the absence of cytochalasin B, it is important that selected test concentrations, based on cytotoxicity, should be similar to what they would have been if cytochalasin B had been used, and should be relevant of a true cytotoxicity. By using models to analyse the dynamics of the cell cultures with and without cytochalasin B we have compared different methods for evaluation of cytotoxicity, and demonstrate that relative decrease in population doubling or relative increase in cell counts are the most appropriate measures of cytotoxicity to compare with reduction in CBPI or RI. 相似文献
7.
Arsenic, widely distributed in the environment, is a potent human carcinogen. Arsenite genotoxicity has been observed in a variety of cells and animal systems. However, the underlying mechanism is not completely clear. In this study, human fibroblasts (HFW) were treated with 1.25-10 microM arsenite for 24 h (low dose and long exposure) and 5-80 microM for 4 h (high dose and short exposure), and the arsenite accumulation, cytotoxicity, and micronucleus (MN) induction were examined. By these two different protocols, HFW cells showed equivalent levels of arsenite accumulation, but exhibited different kinetics of cell killing and different types of MN generation. Arsenite induced mainly kinetochore-positive MN (K+-MN) in HFW cells by low dose exposure whereas mainly kinetochore-negative MN (K--MN) was induced by high dose exposure. Catalase reduced both K+- and K--MN induced by these two exposure protocols. Except for the case of K+-MN induction by the high dose exposure protocol, N-acetyl-cysteine (NAC) in both low and high dose protocols was also shown to effectively reduce arsenite-induced MN. The present results imply that oxidative stress is involved in arsenite-induced MN in diploid human fibroblasts. However, different protocols for arsenite exposure may result in different cellular damage. 相似文献
8.
M. N. Murg M. Schuler D. A. Eastmond 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》1999,446(2):12
Multicolor fluorescence in situ hybridization (FISH) with tandem-labeling probes for the 1cen-q12 region is a potential biomarker for the detection of structural chromosomal aberrations (CAs) in human cells. To determine the suitability of this technique for biomonitoring humans exposed to 1,3-butadiene (BD) and to characterize the alterations induced as well as their stability over time, the human lymphoblastoid cell line AZH-1 was treated with 5 μM diepoxybutane (DEB) or the positive control mitomycin C (MMC; 0.1 μM) for 24 h. Following the removal of the test chemicals, cell cultures were grown for an additional 19 days in the absence of the test compound. Using the tandem FISH technique, aliquots from the main cultures were examined for the induction of CAs affecting the 1cen-q12 region at various intervals. A significant increase in chromosomal breakage/exchanges affecting the 1cen-q12 region was seen in both the DEB- and MMC-treated interphase and metaphase cells. The damage peaked at approximately 48 h following the addition of the test compound and declined with time. However, at day 20, the frequency of aberrant cells was still significantly higher than the control levels. For comparison, the frequency of micronuclei (MN) formed and their origin was determined using the cytochalasin B-modified MN assay and FISH with a pancentromeric probe. Showing a similar pattern, the frequency of centronere-negative MN peaked at 48 h, but however was not significantly elevated above control levels at 20 days. At early time points, aberrations detected using the FISH assay consisted of nearly equal proportions of unstable- and stable-type aberrations, while at the later time points, translocations were the predominant aberration type. In addition, the use of tandem-label FISH in combination with BrdU-immunfluorescence staining, showed that almost identical frequencies of structural aberrations could be seen in actively replicating and non-replicating cell populations. These studies indicate that a small but significant proportion of the alterations detected using this FISH technique persists over time and that this technique may be valuable for biomonitoring chromosomal alterations in BD-exposed populations. 相似文献
9.
The most commonly used genotoxicity assays for cultured mammalian cells are mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell transformation. Since their inception, protocols for these assays have been modified in various laboratories. It has been observed that minor but potentially significant method modifications frequently remain unpublished (Swierenga et al., 1983) but should be considered in the development of recommended protocols. The present study was undertaken to determine the current 'state of the art' for these tests. Detailed questionnaires on culture conditions and testing protocols for both stock and test cell populations were designed with the assistance of an international advisory committee and sent to all research and contract laboratories that could be readily identified in Canada, U.S.A. and Europe. Responses from 425 completed questionnaires were analyzed to determine the most commonly used approach and modifications for each procedural step. As expected, the results show a large degree of interlaboratory variation. Detailed protocols for conducting each assay have been prepared and include: stepwise instructions, precautionary measures and practical solutions to common problems associated with each assay; recipes for media and solutions; formulas for quantifying genotoxic responses; reference lists of related assays; guidelines for interpretation; and discussions of the applications, advantages and disadvantages of each test. 相似文献
10.
Use of the cytokinesis-block micronucleus assay to measure radiation-induced chromosome damage in lymphoblastoid cell lines 总被引:3,自引:0,他引:3
To establish the optimal experimental conditions for the use of the micronuclei (MN) test to determine the level of chromosomal damage induced by ionising radiation (IR) exposure in lymphoblastoid cell lines, a time-course study was performed comparing a normal and an ataxia telangiectasia (AT) cell line, the latter being characterised by an extreme radiation sensitivity. Several parameters were analysed: the use of cytochalasin-B (Cyt-B) to quantify MN, the optimum fixation time to measure radiation-induced MN, the most appropriate treatment dose of IR to distinguish between the normal and the radiosensitive cells and the cell-cycle distribution after irradiation. The results obtained showed that the spontaneous as well as the radiation-induced levels of MN were significantly higher in the AT cell compared to the normal cells (P < 0.001 and P = 0.005, respectively). In both cell types the number of radiation-induced MN were lower in the cultures without Cyt-B than those with Cyt-B (P < 0.001), with the AT cells being distinguished in terms of IR-induced MN from the normal cells only with the addition of Cyt-B. The level of MN formation was independent of the dose of Cyt-B used (3 or 6 microg/ml). The optimum time for radiation-induced MN measured was found to be between 48 and 72 h post-irradiation, with 2 and 4 Gy exposures inducing similar levels of MN. However, as the higher dose caused a greater delay in the cell-cycle, treatment with 2 Gy with MN measurement at 48 h in the presence of 3 microg/ml Cyt-B were chosen as the optimum experimental conditions. This choice was validated using two additional normal and AT cell lines. In conclusion, our results show that the use of Cyt-B increases the sensitivity of the MN test for comparing differences in radiosensitivity between lymphoblastoid cell lines. 相似文献
11.
Adel Courdi Danielle Mari Joël Hérault Pierre Chauvel 《Radiation and environmental biophysics》1995,34(2):85-89
Little is known about the action of charged particles of very high linear energy transfer (LET) on human cells and, in particular, the relationship between DNA damage and reproductive death. The aim of this study was to measure the biological efficiency of a low-energy argon beam (E=7.1 MeV/nucleon, LET= 1590 keV/µm) produced at GSI, Darmstadt, on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colonyforming assay. The MN test, using the cytochalasin-block method, is a measure of genotoxic damage. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colonyforming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 µm2; it was 39 µm2 for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively, at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN. However, multiple (>2) MN in BNC were more frequently observed after low-dose Ar irradiation than after gamma-ray exposure (RBE>1). Moreover, the frequency of multiple MN induction exceeded that expected from a Poisson distribution attribution at all dose levels of Ar irradiation. These results indicate that (1) cell killing after 7.1-MeV Ar beam irradiation is less effective than after Co irradiation at an equivalent average energy deposition; (2) unlike gamma-rays, Ar particles are more efficient at cell killing than in producing MN; (3) the frequent scoring of multiple MN suggests the production of multiple damage sites, even at low fluences of Ar particles. 相似文献
12.
Sai-Mei Hou Fred J. Van Dam Frans de Zwart Caroline Warnock Maddalena Mognato Joanne Turner Natalia Podlutskaja Andrej Podlutsky Roswitha Becker Yvonne Barnett Christopher R. Barnett Lucia Celotti Margaret Davies Edith Hüttner Bo Lambert A. D. Tates 《Mutation research》1999,431(2):21
The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations. 相似文献
13.
Hou SM Van Dam FJ de Zwart F Warnock C Mognato M Turner J Podlutskaja N Podlutsky A Becker R Barnett Y Barnett CR Celotti L Davies M Hüttner E Lambert B Tates AD 《Mutation research》1999,431(2):211-221
The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations. 相似文献
14.
Ishikawa H Ishikawa T Miyatsu Y Kurihara K Fukao A Yokoyama K 《Mutation research》2006,599(1-2):135-143
The cytogenetic effects of cigarette smoke has been evaluated as one of many potential confounders in a large number of biomonitoring studies of occupationally or environmentally exposed populations and control subjects. Despite the well-known presence of carcinogens in the cigarette smoke, the results in the scientific literature linking smoking habits to micronuclei (MN) frequency, one of the cytogenetic markers, are rather controversial. Here, we investigated the relationships among MN frequency, smoking habits and five folate metabolic enzyme gene polymorphisms (MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TYMS 3'UTR) in 132 healthy Japanese men who were non-habitual drinkers. In never- and former-smokers, no statistically significant differences in the mean MN frequencies were observed according to the five folate metabolic enzyme gene polymorphisms. In current-smokers, however, subjects with the AA genotype for MTRR had a significantly higher mean MN frequency than the AG genotypes for MTRR (p<0.05). Furthermore, among subjects with the AA genotype for MTRR, current-smokers were found to have a significantly higher mean MN frequency than never- and former-smokers (p<0.05). To further characterize this association, we stratified the smoking status into five groups: non-smokers (never-smokers and former-smokers), 1-10 cigarettes/day, 11-20 cigarettes/day, 21-30 cigarettes/day and >or=31 cigarettes/day. There was an overall trend for the mean MN frequency in subjects with the MTRR AA genotype to increase as the number of cigarettes smoked per day increased (p<0.01, Jonckheere-Terpstra test). The results of our preliminary study suggest that the MTRR AA genotype acts to increase the MN frequency resulting from cigarette smoking. Therefore, studies on human genotoxicity based on cytogenetic markers of MN should take into account both the MTRR polymorphism and the potential confounding effect of smoking, although these preliminary findings need to be validated in larger populations because of the relatively small sample size. 相似文献
15.
In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances 总被引:1,自引:0,他引:1
von der Hude W Kalweit S Engelhardt G McKiernan S Kasper P Slacik-Erben R Miltenburger HG Honarvar N Fahrig R Görlitz B Albertini S Kirchner S Utesch D Pötter-Locher F Stopper H Madle S 《Mutation research》2000,468(2):137-163
A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro. 相似文献
16.
Helene Filbert Sebastian Attig Nicole Bidmon Bernhard Y. Renard Sylvia Janetzki Ugur Sahin Marij J. P. Welters Christian Ottensmeier Sjoerd H. van der Burg Cécile Gouttefangeas Cedrik M. Britten 《Cancer immunology, immunotherapy : CII》2013,62(4):615-627
Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis. 相似文献
17.
《Mutation Research/Environmental Mutagenesis and Related Subjects》1994,312(1):39-50
The micronucleus (MN) assay in human exfoliated cells has been widely used to detect the genotoxic effects of environmental mutagens, infectious agents and heriditary diseases. Substantial variability characterizes the MN frequencies reported by different research groups. One reason for this may be the restricted resolution power of the Feulgen-Fast-Green staining that is routinely used. Here we describe a new version of the MN assay that employs fluorescent propidium iodide staining along with fluorescence in situ hybridization (FISH) with a centromeric probe. Buccal and urothelial cells were collected from 5 healthy unexposed female volunteers and 55 000 cells analyzed for MN frequency and abnormal nuclear events. The Feulgen-Fast-Green and the new fluorescent staining produced very similar results. The frequency of MN in buccal cells was 0.145±0.118% and in urothelial cells 0.083±0.074%. No correlation was found between the frequencies of MN in the two types of exfoliated cells. FISH with a centrometric probe allowed MN containing whole chromosomes with a centromere to be differentiated from those containing only acentric fragments. The former appear as a result of chromosome lagging in mitosis, while those without a centromere are due to chromosome breakage. In urothelial cells 43% of MN were centromere-negative and in buccal cells — 44%. Fluorescent staining provided more accurate scoring of degenerative cells than standard Feulgen-Fast-Green staining. The combined frequency of pycnotic cells, “broken eggs” and cells with fragmented nuclei did not exceed 2%, while that of karyorrhexis and karyolysis together was as high as 21%. Significant interindividual variability was found in the frequency of cells with karyolysis and karyorrhexis. Thus, the new version of micronucleus assay allows for MN to be scored more precisely, the mechanism of MN formation to be determined and abnormal nuclear events to be readily identified in exfoliated human cells. It is therefore ideal for studying genotoxicity in human populations using exfoliated cells from the mouth, bladder and nose. 相似文献
18.
The formation of micronuclei (MN) is extensively used in molecular epidemiology as a biomarker of chromosomal damage, genome instability, and eventually of cancer risk. The occurrence of MN represents an integrated response to chromosome-instability phenotypes and altered cellular viabilities caused by genetic defects and/or exogenous exposures to genotoxic agents. The present article reviews human population studies addressing the relationship between genetic polymorphisms and MN formation, and provides insight into how genetic variants could modulate the effect of environmental exposures to genotoxic agents, host factors (gender, age), lifestyle characteristics (smoking, alcohol, folate), and diseases (coronary artery disease, cancer). Seventy-two studies measuring MN frequency either in peripheral blood lymphocytes or exfoliated cells were retrieved after an extensive search of the MedLine/PubMed database. The effect of genetic polymorphisms on MN formation is complex, influenced to a different extent by several polymorphisms of proteins or enzymes involved in xenobiotic metabolism, DNA repair proteins, and folate-metabolism enzymes. This heterogeneity reflects the presence of multiple external and internal exposures, and the large number of chromosomal alterations eventually resulting in MN formation. Polymorphisms of EPHX, GSTT1, and GSTM1 are of special importance in modulating the frequency of chromosomal damage in individuals exposed to genotoxic agents and in unexposed populations. Variants of ALDH2 genes are consistently associated with MN formation induced by alcohol drinking. Carriers of BRCA1 and BRCA2 mutations (with or without breast cancer) show enhanced sensitivity to clastogens. Some evidence further suggests that DNA repair (XRCC1 and XRCC3) and folate-metabolism genes (MTHFR) also influence MN formation. As some of the findings are based on relatively small numbers of subjects, larger scale studies are required that include scoring of additional endpoints (e.g., MN in combination with fluorescent in situ hybridization, analysis of nucleoplasmic bridges and nuclear buds), and address gene-gene interactions. 相似文献
19.
Yellow Fever Virus: I. Development and Evaluation of a Plaque Neutralization Test 总被引:6,自引:6,他引:0
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Heretofore, the most reliable way of measuring yellow fever virus antibody was to use the mouse neutralization (MN) test employing either suckling or weanling mice. Certain disadvantages (e.g., expense both of animals and of maintaining a mouse colony, allergic reactions of many laboratory workers, and the relatively long time, 21 days, before end points are reached) are inherent in any program with mice or other laboratory animal species and have discouraged the use of the MN test by many laboratories. A previously reported plaque neutralization (PN) test with primary chick embryo cell cultures could not be consistently reproduced by later investigators. We have developed a convenient and reproducible PN test employing the MA-104 embryonic rhesus monkey kidney cell culture and a single agar-overlay procedure. When compared with MN tests with newborn (1 to 3 days old) and weanling (16 to 20 g, 24 to 28 days old) mice inoculated by the intracranial route, the PN test was the most sensitive for measuring neutralizing antibody; it was also less variable, less costly, and it achieved results in the shortest period of time. End points could be determined in 5 to 6 days for the PN test as compared to 21 days for the MN test. 相似文献
20.
Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1–1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects. Bioelectromagnetics 19:85–91, 1998. © 1998 Wiley-Liss, Inc. 相似文献