Linked Life Data

16180205

Source:http://linkedlifedata.com/resource/pubmed/id/16180205

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2006-1-12
pubmed:abstractText
The in vitro micronucleus test has received considerable attention in recent years for its use in drug safety assessment and toxicological research. The less tedious nature of the assay relative to chromosome aberration analyses is a driving force, and explains why many chemical and drug safety programs have adopted the endpoint. Development of a high-throughput micronucleus scoring system would further enhance the utility of the assay for lead optimization and other early drug development work. Although several variations of a flow cytometric (FCM) method for scoring cell-culture-derived micronuclei (MN) have been described in the literature, they have been unable to distinguish true MN from apoptotic and necrotic chromatin (Nüsse M and Marx K 1997: Mutat Res 392: 109-115). Here, we report advances to this methodology whereby a sequential staining procedure is used to differentially label these types of sub-2n particles. With the use of ethidium monoazide (EMA), the chromatin of dead and dying cells is labeled. After a photoactivation step that covalently binds EMA to chromatin, cytoplasmic membranes are digested and resulting lysates are incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and sub-2n particles that are labeled with either SYTOX or SYTOX and EMA. Preliminary studies with heat-shocked L5178Y mouse cells demonstrated that EMA stains necrotic and mid- through late-stage apoptotic cells. Importantly, the sequential labeling procedure provided reliable micronucleus enumeration, even when cultures contained high percentages of dead cells. Subsequently, experiments with the following diverse genotoxicants were performed: hydroxyurea, methyl methanesulfonate, benzo[a]pyrene, etoposide, cyclophosphamide, and vinblastine. Additionally, the nongenotoxicants sucrose, tributyltin methoxide, and dexamethasone were tested up to 5 mg/ml, or to cytotoxic concentrations. FCM data were found to correspond closely with microscopy-based measurements. Collectively, these data suggest that this sequential EMA/SYTOX staining procedure provides reliable, high-throughput enumeration of in vitro MN.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0893-6692
pubmed:author
pubmed:copyrightInfo
2005 Wiley-Liss, Inc.
pubmed:issnType
Print
pubmed:volume
47
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
56-66
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability.
pubmed:affiliation
Litron Laboratories, Rochester, New York, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural