In our study, the freshwater bivalve unio pictorum was selected as test organism. The use of a multimarker approach coupling genotoxicity biomarkers with physiological and biochemical factors allows to have a complete picture of the environmental pollution current zoology 60 2. During acclimatisation, they were kept in a flowthrough aquarium at the facility of the balaton limnological research centre. Oxidative dna damage may not mediate niinduced genotoxicity. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a largescale radiologicalnuclear event. The micronucleus assay in toxicology is the first comprehensive volume concerning the use of micronucleus assays in genetic toxicology. Assessment of the genotoxicity and cytotoxicity in. In the micronucleus test, unio pictorum painters mussel specimens with length of 58 cm were used. This protocol describes one of the current established methods for buccal cell collection using a.
Oecd 2004 oecd guideline for the testing of chemicals draft proposals for a new guideline 487. Keywords aquatic animals, genotoxicity, bioindicator, biomarker, micronucleus test, comet assay, bivalve, mussel. The mussel mn cytome mumncyt assay protocol we describe here reports the recommended experimental design, sample size, cell. Application of the micronucleus and comet assays to mussel. Several studies have provided information on environmental nanoplastic particlesdebris, but the in vitro cytogenotoxicity is still insufficiently characterized. Caged mussels, collected from the river drava, were transplanted to four monitoring sites of different pollution intensity in the river sava. A micronucleus test is a test used in toxicological screening for potential genotoxic compounds.
The cytokinesisblock micronucleus cbmn assay was first developed to score micronuclei in cells that completed nuclear division by blocking them at the binucleate stage before cytokinesis. Imaging flow cytometry ifc, which combines the statistical power of traditional flow cytometry with. Lpiccp protocol for in vitro micronucleus assay using chok1 cells. The title of the article insensitivity of the in vitro cytokinesisblock micronucleus assay with human lymphocytes for the detection of dna damage present at the start of the cell culture is incorrect and misleading because there is overwhelming evidence that the cbmn assay is highly sensitive to ionising radiation, radiomimetic compounds. Pdf the micronucleus assay as a biological dosimeter of. A total of 20 slides 10 for cases and 10 for controls were reexamined a few months after the first measurement. Histological changes and micronucleus induction in the zebra.
Immunocytotoxicity, cytogenotoxicity and genotoxicity of. The nuclear abnormality assay did not appear to be an efficient tool for genotoxicity evaluation when compared with field samples taken at a reference site in laboratory, with a positive control. In vivo rodent micronucleus assay has been widely used to detect genotoxicity. It offers great opportunity to evaluate genotoxicity by the way of quantifying mean frequencies of micronuclei, binucleated cell, broken egg, karyolysis, karyorrhexis, pycknosis and condensed. Micronucleus assay pdf the in vitro micronucleus assay is a mutagenic test system for the detection medical electronics pdf of. The aim of the present study was to estimate levels of mi.
A highthroughput in vivo micronucleus assay for genome. Buccal micronucleus cytome assay was used to test for biomarkers of dna damage, cell death proliferation, and cell death in both sle patients and controls. Bioreliance offers the most comprehensive portfolio of assays and designs to meet all regulations and product types requiring mutagenicity or genetic toxicology testing. The cytokinesisblock micronucleus cbmn assay this technique consists in adding to cell cultures cytochalasinb cytb, an inhibitor of the mitotic spindle that prevents cytokinesis. Nanomaterials free fulltext in vitro genotoxicity of. Effectiveness of the liver micronucleus assay using juvenile mice.
The micronucleus assay due to its potentiality to be ap. The cytokinesisblock micronucleus cytome assay cbmncyt in peripheral blood lymphocytes is the most frequent method in biomonitoring human populations to evaluate exposure to genotoxic agents, micronutrient deficiency, or excess and genetic instability. The cytokinesisblock micronucleus cytome assay in peripheral blood lymphocytes lcbmncyt is the most frequently used method in biomonitoring human populations to evaluate dna damage caused by exposure to genotoxic agents, micronutrient. Comet and micronucleus assays in zebra mussel cells for. These micronuclei may originate from acentric fragments chromosome fragments. The test is based on the formation of micronuclei which indicates chromosomal dna damage occurring as a result of either chromosome breakage or chromosome missegregation during mitosis. Short communication testing chemical agents with the. In addition, the mechanism of micronuclei formation is discussed. It is an easytoperform test which has proven sensitive to tannins or tannin containing extracts. The cytokinesisblock micronucleus cbmn assay tomisato miura, ph. Washing soda induced alteration of the differential cell. Micronucleus assay eight mussels from each experimental group were sampled for the micronucleus assay mn after 7 and 14 days of exposure. Oct 01, 2014 read immunocytotoxicity, cytogenotoxicity and genotoxicity of cadmiumbased quantum dots in the marine mussel mytilus galloprovincialis, marine environmental research on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.
The buccal micronucleus cytome bmncyt assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and followup of precancerous oral lesions. Concerns about the adverse health effects of chemicals and radiation present in the environment and at workplaces have created the need for better detection systems to assess their potential to cause dna damage in humans and other organisms across ecosystems. The use of fish is limited to specific geographic areas. Pdf application of the buccal micronucleus cytome assay. Genotoxicity biomarkers in aquatic bioindicators semantic. We evaluated the genotoxic effects detected in different tissues haemocytes for the comet assay and gills for the micronucleus test mn of the filterfeeder mussel dreissena polymorpha in vivo exposed in experimental basins supplied with lake trasimeno water withwithout the different disinfectants during seasonal periods in the year. Mussels were collected in lake balaton hungary and were acclimatized for 1 month prior to the test. The application of the cytokinesisblock micronucleus assay on peripheral blood lymphocytes for the assessment of genome damage in longterm residents of areas with high radon concentration. Background in general, the term genotoxicity refers to the ability to induce structural changes in genes via. In vitro cytogenotoxic evaluation of sertraline in. Nowadays, the human buccal micronucleus cytome bmcyt assay is one of the most widely used techniques to measure genetic damage in studies on human population exposed to airborne pollutants 19,20.
Evaluation of genotoxicity using the micronucleus assay and. Cytokinesisblock micronucleus cbmn assaycbmn cytome. A buccal cytome assay was used to measure ratios of buccal cell populations and micronuclei in clinically diagnosed alzheimers patients compared to age and gendermatched controls. We use cookies to offer you a better experience, personalize content, tailor advertising, provide social media features, and better understand the use of our services. The mn formation in the cytoplasm of cells that have undergone proliferation is a consequence of dna fragmentation during mitosis and the appearance of small additional nuclei during interphase.
Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology galina g hovhannisyan abstract comet assay and micronucleus mn test are widely applied in genotoxicity testing and biomonitoring. The micronucleus testmost widely used in vivo genotoxicity test. We tested the cytokinesisblock micronucleus cytome assay using the wil2ns human b lymphoblastoid cell line as a biomarker of genotoxicity and cytotoxicity of cecal water from rats and fecal water from humans. Treatment of sublethal concentrations of washing soda for 384 hours exhibited a significant decrease in the densities of blast like cells, small and large amoebocytes. Pdf aquatic pollutants produce multiple consequences at organism. The aim of this study is to analyze the effects of polystyrene nanoparticles pnps in the hs27 cell line. Cytokinesisblock micronucleus cytome assays for the.
The micronucleus mn assay is a method currently used as an alternative and simple means of assessing. Apart from the evaluation of micronuclei, the cbmn cytome assay allows to assess other relevant. The micronucleus mn assay in human exfoliated cells. It later evolved into the cbmn cytome assay to further explore cell death, cytostasis, and biomarkers of dna damage. Alzheimers disease ad is a progressive degenerative disorder of the brain and is the commonest form of dementia. Micronucleus cytome assay in the differential assessment. The buccal micronucleus cytome assay in exfoliated buccal cells is utilized as biomarkers for dna damage, cell death and basal cell frequency. While comet assay permits to measure direct dnastrand breaking capacity of a tested agent mn test. Cytokinesisblock micronucleus assay evolves into a cytome assay of chromosomal instability, mitotic dysfunction and cell death.
Washing soda has been identified as a precarious contaminant of the freshwater ponds and lakes, the natural habitat of eunapius carteri. Micronucleus cytome mncyt assay, a comprehensive cytogenetic test for the assessment of genome instability induced by xenobiotics in organisms, was utilized in the differential cytogenotoxic evaluation of cd and pb in adult amietophrynus regularis. Pdf the cytokinesisblock micronucleus cytome cbmn cyt assay is a new and comprehensive technique for measuring dna damage. Biological dosimetry, based on the analysis of micronuclei mn in the cytokinesisblock micronucleus cbmn assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. The main limitation for its use is the lack of knowledge about inter and intralaboratory variability in scoring micronuclei and other end points included in. Bolognesi c, fenech m 2012 mussel micronucleus cytome assay. Lythrum salicaria extract determined by the mussel. The micronucleus assay in toxicology rsc publishing. Because the mussel mn assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an mn cytome assay. Interlaboratory consistency and variability in the buccal. Jun 14, 20 read oxidative dna damage may not mediate niinduced genotoxicity in marine mussels. Read comet and micronucleus assays in zebra mussel cells for genotoxicity assessment of surface drinking water treated with three different disinfectants, science of the total environment on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Dna damage events are scored specifically in oncedivided.
The bone marrow micronucleus test may be considered as a method for aneuploidy detection. Evaluation of micronucleus induction is the primary in vivo test in a battery of genotoxicity tests and is recommended by the regulatory agencies around the globe to be conducted as part of product safety assessment. Over the past few years, the cbmn test has evolved into a more comprehensive method for measuring dna damage, cytostasis. Dna damage and cytotoxicity biomarkers in the lymphocyte cytokinesisblock micronucleus cytome cbmn cyt assay. Assessment of dna damage is of primary concern when determining the pollutionrelated stress in living organisms.
This study investigated the effectiveness of the liver micronucleus mn assay using juvenile mice. The mussel mncytome mumncyt assay, using the proposed detailed criteria for the identification of cell. The in vitro cytokinesisblock micronucleus cbmn assay is a cytogenetic method based on the assessment of micronuclei in nucleated cells that have completed only one nuclear division 4, 5. Reliability of measurements was assessed by intraexaminer calibration. In summary, the highthroughput micronucleus assay is a fast and sensitive method for the detection of micronuclei, but further experiments are needed to ascertain the mechanism of its formation. Micronucleus test good biomarker for determination of genetic changes in aquatic organism. The viability of hs27 cells was determined following exposure at different time windows and pnp concentrations. However, some extracts were slightly mutagenic in the comet assay and this may be attributed to cytotoxicity rather than genotoxic effects. No plant extract was mutagenic in the ames test and micronucleus cytome assay. The in vitro micronucleus test provides an alterative to the chromosome aberration test, and because the in vitro micronucleus test examines cells at interphase, the assessment of micronuclei can be scored faster, as the analysis of damage is thought to be less subjective and is more amenable to automation. Cecal water was assessed in rats fed either a diet rich in fat, low in calcium and fiber, and barbecued red meat as the protein source high colorectal cancer risk diet or a diet high. The upper edge of the mussel shells was gently broken and about 100 l haemolymph were drawn up from the sinus near the posterior adductor muscle by a hypodermic syringe. The mussel micronucleus test is a widely established, relatively easytoperform test.
Cyt assay was developed as a system for evaluating dna damage, cytostasis, and cytotoxicity. Pdf micronucleus assay in aquatic animals researchgate. A standard harmonized battery of genetic toxicology assays is an integral part of many global regulatory guidelines. The author believes the micronucleus test as a related to but an independent method from chromosomal aberration test with its own characteristics. The in vitro micronucleus assay is a mutagenicity test system used for the detection of chemicals that induce the formation of small membranebound dna fragments such as micronuclei in the cytoplasm of interphase cells. As a consequence, cells that have completed one nuclear division are identified by their binucleated appearance. The micronucleus cytome assay applied in buccal exfoliated cells bmncyt. Micronuclei formation indicates chromosomal dna damage occurring as a result of either chromosome breakage or chromosome missegregation during mitosis 1. Both the ames test and the micronucleus test showed clear concentrationresponse pattern, however, the ames test. To monitor genotoxicity of the freshwater environments we used micronucleus mn and comet assay on dreissena polymorpha haemocytes. Applications of the cytokinesisblock micronucleus cytome. N2 the micronucleus mn assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal dna damage occurring as a result of either chromosome breakage or chromosome missegregation during mitosis.
The cytokinesisblock micronucleus cytome cbmn cyt assay was employed to count the frequencies of micronuclei and mononucleated cells per binucleated cell in blood samples from 10 healthy donors, using manual scoring to collect micronuclei data and an automated program to collect. Micronucleus cytome assays in human lymphocytes and buccal. Micronucleus cytome assay in the differential assessment of. Reevaluation of the cytokinesisblock micronucleus assay using semiautomated scoring following telomere and centromere staining. Mussels, due to their widely geographical distribution and the suitability for caging represent the bioindicator of choice in field studies. Genotoxic effect in autoimmune diseases evaluated by the.
Buccal micronucleus cytome biomarkers may be associated. Chapter 17 the micronucleus assay in toxicology rsc. Genotoxicity biomarkers in the assessment of heavy metal. Biological dosimetry or biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Increased micronuclei frequency in oral and lingual. A standardized mn assay protocol in hemocytes and gill cells for use in bivalve species, including scoring of different cell types, necrotic and apoptotic cells and nuclear anomalies, was established following the cytome approach. Treatment of sublethal concentrations of washing soda for 384 hours exhibited a significant decrease in the densities of. Pdf buccal micronucleus cytome assay michael fenech. Assessment of genotoxic biomarkers and transcriptional responses of key stress genes, mutation research genetic toxicology and environmental mutagenesis on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. May 17, 2012 because the mussel mn assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an mn cytome assay. The buccal micronucleus cytome bmcyt assay is a minimally invasive method for studying dna. Buccal micronucleus cytome assay springer nature experiments. The cytokinesisblock micronucleus cytome assay is a comprehensive system for measuring dna damage, cytostasis and cytotoxicity. Oct 01, 2016 however, the micronucleus test is not suggested to be a simply alternative method for metaphase analysis or the results of the assay have less value compared to those of chromosomal aberration test.
Micronucleus frequency in exfoliated buccal cells of. The assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens, i. In the present study, the genotoxic potential of srt was evaluated using cytokinesisblock micronucleus cbmn cytome assay in peripheral blood lymphocytes of healthy human subjects. The mussel mn cytome mumncyt assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining. Following micronucleus scoring criterion, score binucleated. Fluorescence in situ hybridization in combination with the. The in vitro inhibition of genotoxicity by plant extracts. Therefore, we analyzed various hepatic cytochrome p450 cyp mediated activities of ethoxyresorufin odeethylation, pentoxyresorufin odealkylation, tolbutamide hydroxylation, bufuralol 1hydroxylation, aniline hydroxylation and midazolam 4hydroxylation by cyp1a, cyp2b, cyp2c, cyp2d, cyp2e. Application of the buccal micronucleus cytome assay and analysis of pon1gln192arg and cyp2a69. Cytome protocol including the identification of different cell types and. Assessing genotoxic potential of cotinus coggygria scop. Cytokinesisblock micronucleus cytome assay nature protocols. The buccal micronucleus cytome bmcyt assay is a minimally invasive approach for measuring dna damage, cell proliferation, cell differentiation and cell death in exfoliated buccal cells. Sanchezparada, buccal micronucleus cytome assay of populations under chronic heavy metal and other metal exposure along the santiago river, mexico, environmental monitoring and assessment, 189, 10, 2017.
Micronucleus test good biomarker for determination of. In the second protocol, mn test is applied on gill cells according to the procedure proposed by. Micronucleus mn assay can be performed in order to evaluate loss of genetic material in a clear, accurate, fast, simple, and minimally invasive test. Dolcetti l, venier p 2002 susceptibility to genetic damage and cell types in mediterranean mussels. Micronucleus cytome assay is a technique used to determine smaller than normal nucleus and other nuclear abnormalities resulting from the incorrect splitting and sharing of genetic material replicated during mitosis due to external insults such as ionizing radiation or genotoxic substances. The protocol for lcbmncyt described here, refers to the use of ex vivo whole. Different aspects of cytochalasin b blocked micronucleus cytome. In this work, micronuclei were induced by two aneugens, vincristine sulfate 0. Srt blocks serotonin reuptake and increases serotonin stimulation of somatodendritic serotonin 1a receptor 5ht1ar and terminal autoreceptors in the brain. The cytokinesisblock micronucleus cbmn assay has become a fullyvalidated and standardized method for radiation biodosimetry. Labieniec m, gabryelak t, falcioni g 2003 antioxidant and prooxidant effects of tannins in digestive cells of the freshwater mussel unio tumidus. The micronucleus test should in most studies be accompanied by evaluation of the cytochalasinb proliferation index or the nuclear division index by counting the frequency of mono, bi, tri and tetranucleated cells.
893 1035 884 1235 18 1592 289 1413 442 1478 194 1392 702 226 939 226 1490 137 280 161 26 895 1167 36 39 554 721 1113 1268 1260 1134 1309 1486 1099 511 534 1080 704 1318 278 1050 939